Methods of Modifying Pulp Comprising Cellulase Enzymes and Products Thereof

ABSTRACT

Provided are methods for preparing an enzyme-modified fiber pulp, wherein the modified pulp has improved properties for papermaking processes. The modified pulp and methods of its use are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of the filing date of U.S. Provisional Application No. 62/395,698, filed Sep. 16, 2016.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 13, 2017, is named 205666_5069_WO_566976_ST25.txt and is 48,542 bytes in size.

BACKGROUND

In the discussion of the background that follows, reference is made to certain structures and/or methods. However, the following references should not be construed as an admission that these structures and/or methods constitute prior art. Applicants expressly reserve the right to demonstrate that such structures and/or methods do not qualify as prior art.

It has been well established knowledge as well as commercial practice, that cellulose enzymes can be used in pulp and paper applications, such as drainage improvement, reducing refining energy, de-inking, re-activating the recycled fiber furnish, and under certain circumstances enhancing tensile strength. See, for example, U.S. Pat. No. 4,923,565 (Fuentes et al.), EP 0583310 (Leclerc), and U.S. Pat. No. 5,169,497 (Sarkar et al.). The use of cellulase treatment in combination with various papermaking agents or polymers has also been reported. These uses have various name designations for the agents or polymers used, such as dry strength agents, wet strength agents, retention agents, flocculation agents, fixing agents, de-contaminating agents, and enzyme stabilizers. These agents include various cationic polymers, anionic polymers, nonionic polymers, amphoteric polymers, and various anionic and nonionic surfactants. See, for instance, U.S. Pat. No. 5,169,497 (Sarkar et al), U.S. Pat. Nos. 5,423,946, 5,507,914, U.S. Pat. No. 6,939,437 (Hill et al.), US Publ. No. 2002-0084046 (Hsu et al.), U.S. Pat. No. 6,066,233 (Olsen et al.), U.S. Pat. No. 6,770,170 (Covarrubias), US Publn. No. 2011-0168344 (Klein et al.), US Publn. No. 2014-0116635 (Porto et al.), and U.S. Pat. No. 9,011,643 (Gu et al.).

It is generally understood by the skilled artisan in the art that cellulase enzymes, sometimes in combination with proper hemicellulase enzymes related to the furnish to be treated, work to hydrolyze the hydrated fiber surface (“gel”) layers, such as caused by refining or mechanical actions, thus improving drainage and freeness. It is also understood by the skilled artisan that endoglucanases actions of cellulases work on the amorphous regions of the fibers, softening the fibers, for refining. It is also generally recognized that cellulase enzymes, by proper formulations or dosing, may work on the fiber surface, causing external fiber surface fibrillations, therefore improving bonding or tensile strength. The skilled artisan recognizes that the cellulase treatment can cause interior damage to the fiber which adversely affects fiber strength properties and fiber length reductions. Consequently, in the application of cellulase enzymes to wood fiber for papermaking, care is exercised not to cause interior damage to the fiber, and therefore preserve fiber strength and length.

Mechanical pulping consumes enormous amount of electrical energy for defiberization and pulp fiber development. Various strategies have been used or proposed to reduce the electrical energy requirements. Traditional commercial practices include SO₂ or bisulfite additions (e.g., CTMP), alkaline peroxide additions (e.g., APMP), high temperature and duration treatment (e.g., Thermo-pulp), steam explosions, and cold-soda pretreatment. On the research investigations side, various chemical pretreatment have been evaluated. For instance, a catalytic oxygenation treatment based on vanadyl organic complexes or vanadyl sulfate, for instance, in the impressifiner stage or a coarsely refined stage including secondary rejects for mechanical pulping, wherein 50% energy reduction and substantially improved fiber conformability has been reported. See, e.g., WO 1998020199.

There is an on-going unmet need in the art for fiber pulps having improved properties for papermaking and other products. The present disclosure addresses this need.

SUMMARY

The following summary is not an extensive overview. It is intended to neither identify key or critical elements of the various embodiments, nor delineate their scope.

The present disclosure is directed to methods of preparing modified pulp. The method can intentionally inducing a controlled degree of fiber interior damage, as exemplified by noted fiber length reduction at given freeness. The resulting fiber is useful in a variety of applications for the manufacturing of paper, pulp, or web products. Specifically, it has been found that by intentionally inducing significant but controlled degree of fiber interior modification or “fiber interior damage” (in addition to fiber surface modifications), as exemplified by a noted fiber length reduction, a variety of papermaking benefit can be exploited in synergy with select papermaking chemicals.

Provided is a method of modifying papermaking pulp comprising contacting pulp comprising papermaking fibers with (a) a cellulolytic enzyme under conditions to induce fiber interior damage, and (b) a fiber length reduction enhancement agent to produce a modified pulp having: (i) a reduction of papermaking fiber length of 5% to 96% after going through a refiner or after by-passing a refiner, and (ii) a pulp freeness level of from about 200 Canadian Standard Freeness (CSF) to about 700 CSF. The modified pulp can be a mechanically refined pulp.

Contact of the pulp with the fiber length reduction enhancement agent preferably occurs after contacting the pulp with the enzyme. Alternatively, contact of the pulp with the fiber length reduction enhancement agent occurs before or at the same time as contacting the pulp with the enzyme.

The method can use a fiber length reduction enhancement agent selected from the group consisting of: a hydrolyzed polymer or copolymer of vinylformamide with a degree of hydrolysis greater than 70%; a copolymer of acrylamide and acryloxyethyl-trimethylammonium chloride; a copolymer of acrylamide and diallyldimethylammonium chloride; a free glyoxal; and any mixture of two of more thereof.

The method can use a cellulolytic enzyme which is a cellulase or a cellulase-functioning hemicellulase enzyme. The cellulolytic enzyme can be a cellulase selected from the group consisting of: a cellulase obtained or derived from Chrysosporium lucknowense/Myceliophthora thermophilia, a cellulase obtained or derived from Humicola insolens, a cellulase obtained or derived from Aspergillus, a cellulase obtained or derived from Trichoderma, and combinations thereof.

The pulp of the method can comprise papermaking fiber selected from the group consisting of: a softwood fiber, a hardwood fiber, and a mixture thereof.

The fiber length reduction enhancement agent of the method can be selected from the group consisting of: a hydrolyzed polymer or copolymer of vinylformamide with a degree of hydrolysis greater than 70% and a copolymer of acrylamide and acryloxyethyl-trimethylammonium chloride; and the reduction of papermaking fiber length of the modified pulp is from about 5% to about 20% and the pulp freeness level of the modified pulp is from about 300 CSF to about 650 CSF.

The papermaking fiber of the method can be a wood fiber and the fiber length reduction enhancement agent is selected from the group consisting of: a hydrolyzed polymer or copolymer of vinylformamide with a degree of hydrolysis greater than 70% and a copolymer of acrylamide and acryloxyethyl-trimethylammonium chloride; and is present at a dosage of from 0.5 kilogram agent per ton pulp (kg/ton pulp) to about 29 kg/ton pulp, wherein the pulp freeness level of the modified pulp is from about 350 CSF to about 650 CSF.

The fiber length reduction enhancement agent of the method can be selected from the group consisting of: a hydrolyzed polymer or copolymer of vinylformamide with a degree of hydrolysis greater than 70% and a copolymer of acrylamide and acryloxyethyl-trimethylammonium chloride; and is present at a dosage of from 0.5 kilogram agent per ton pulp (kg/ton pulp) to about 29 kg/ton pulp, and the reduction of papermaking fiber length of the modified pulp is from about 5% to about 20% and the pulp freeness level of the modified pulp is from about 300 CSF to about 650 CSF.

Also provided is an enzyme-modified fiber pulp of any of the methods described above or herein.

Also provided is a method of modifying pulp fiber comprising contacting pulp comprising papermaking fibers with a thermostable or hyperthermostable cellulolytic enzyme at a temperature from about 70° C. to about 125° C., wherein the cellulase enzyme is substantially inactive at a temperature from about 35° C. to about 55° C. The refining energy of the pulp or fibrous material of the method can be reduced by at least 10%. The modified pulp of the method can have a hemicellulose content that is reduced by at least 0.2 wt. % compared to the hemicellulose content of a pulp made without contacting with the thermostable or hyperthermostable cellulolytic enzyme. The cellulolytic enzyme of the method can be a cellulase, a cellulase-functioning hemicellulase enzyme, or a cellulase containing concomitant hemicellulase enzyme functions such as xylanases, mannanases, glucanases, and beta-glucanases. The cellulolytic enzyme of the method can be a cellulase selected from the group consisting of: cellulase of SEQ ID NO: 6 and cellulase of SEQ ID NO: 2. The contacting step of the method can further comprising a thermostable or hyperthermostable enzyme selected from the group consisting of: a hemicellulase, a mannanase, a xylanase, a pectinase, a laccase, a lignin peroxidase, a manganese peroxidase, and an oxidoreductase. The pulp of the method comprises papermaking fiber selected from the group consisting of: a softwood fiber, a hardwood fiber, and a mixture thereof. The enzyme contacting step of the method can further comprise an intracrystalline lignocellulosic agent selected from the group consisting of an ionic liquid, an N-alkylated urea, an N-alkylated lactam, an N-alkylated amide, a polyol ether, a polyol, and combinations thereof. The pulp of the method comprises a kraft pulp, a sulfite pulp, a mechanical pulp, a semi-chemical pulp, a semi-mechanical pulp, a chemi-mechanical pulp, a fluff pulp, a tissue pulp, a dissolving pulp, a recycled pulp, a biorefinery pulp, or a deinked pulp. Also contemplated is an enzyme-modified fiber pulp produced by the above method.

Also provided is a method of modifying papermaking pulp comprising: (1) contacting pulp comprising softwood fibers having a fiber length of about 2.1 mm to about 2.8 mm, as measured by FQA length-weighted fiber average length, with a cellulolytic enzyme at a temperature of about 35° C. to about 125° C., (2) refining the modified pulp, to produce a modified pulp having: (i) a fiber length of about 0.6 mm to about 1.7 mm, and (ii) a pulp freeness level of from about 400 Canadian Standard Freeness (CSF) to about 700 CSF. The contacting step of the method can be carried out at a temperature of about 35° C. to about 125° C. for about 30 minutes to about 10 hours. Alternatively, the contacting step can be carried out at a temperature of about 35° C. to about 65° C. and at an enzyme dosage of about 0.2 kilogram enzyme per ton of pulp (kg/T) to about 20 kg/T. The cellulolytic enzyme of the method can be a cellulase selected from the group consisting of: a cellulase obtained or derived from Chrysosporium lucknowense/Myceliophthora thermophilia, a cellulase obtained or derived from Humicola insolens, a cellulase obtained or derived from Aspergillus, a cellulase obtained or derived from Trichoderma, and combinations thereof. The contacting step of the method can be carried out at a temperature of about 66° C. to about 125° C. and at an enzyme dosage of from about 0.2 kilogram enzyme per ton of pulp (kg/T) to about 20 kg/T. The cellulolytic enzyme of the method can be a cellulase or a cellulase-functioning hemicellulase enzyme. The cellulolytic enzyme of the method can be the cellulase of SEQ ID NO: 6 or SEQ ID NO: 2. The method can also comprise contacting the pulp with a fiber length reduction enhancement agent. The method wherein contacting the pulp with the fiber length reduction enhancement agent contact occurs preferably after contacting the pulp with the enzyme.

Also provided is an enzyme-modified softwood fiber pulp having: (i) a fiber length of about 0.6 mm to about 1.7 mm, and (ii) a pulp freeness level of from about 400 Canadian Standard Freeness (CSF) to about 700 CSF.

Also described herein is a paper web or pulp product comprising a modified pulp produced by any of the disclosed methods. The paper web or pulp product can be selected from the group consisting of: printing writing papers, packaging papers, paper boards, personal care and hygiene products, paper tissues, paper towels, paper napkins, paper wipes, paper plates, paper cups, paper containers, deinked papers, recycled papers, microcrystalline cellulose, microfibrillated cellulose, nanofibrillated cellulose, nanocrystalline cellulose, dissolving pulp, and base papers for polymer impregnation or base papers for composites.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosed compounds, compositions, and methods.

BRIEF DESCRIPTION OF FIGURES

For the purpose of illustrating the various products, compositions and methods, there are depicted in the drawings certain embodiments. However, the products, compositions, methods of making them, and methods of their use are not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

FIG. 1 depicts a graph plotting freeness (CSF, Canadian Standard Freeness) against PFI mill revolutions for a control pulp and two enzyme-treated pulps.

FIG. 2 depicts a graph tensile index (in NM/g) against PFI mill revolutions for a control pulp and two enzyme-treated pulps.

FIG. 3 depicts a graph plotting Sheffield smoothness (in ml/min) against PFI mill revolutions for a control pulp, two enzyme-treated pulps, and an enzyme-treated pulp subsequently contacted with a fiber length reduction enhancing agent prior to refining.

FIG. 4 depicts a graph plotting Sheffield smoothness (in ml/min) against freeness (CSF) for a control pulp, two enzyme-treated pulps, and an enzyme-treated pulp subsequently contacted with a fiber length reduction enhancing agent prior to refining.

FIG. 5 depicts a graph plotting length-weighted fiber length (LWAFL) against freeness for a control pulp, two enzyme-treated pulps, and an enzyme-treated pulp subsequently contacted with a fiber length reduction enhancing agent prior to refining.

FIG. 6 depicts a graph plotting freeness (CSF, Canadian Standard Freeness) against Valley-Beater refining time (in minutes) for a control pulp and an enzyme-treated pulp. Freeness was measured on the pulp after it was blended with Xelorex™.

FIG. 7 depicts a graph plotting paper bulk (in cc/g) against Valley-beater refining time (in minutes) for DSF (Dynamic Sheet Former) sheets made from the control pulp and the enzyme-treated pulp of FIG. 6. The pulps were blended with Xelorex™ after Valley-Beater refining and made into DSF sheets.

FIG. 8 depicts a graph plotting paper bulk (in cc/g) against Freeness (in CSF) after Valley-Beater refining for DSF (Dynamic Sheet Former) sheets made from the control pulp and the enzyme-treated pulp of FIG. 6. The pulps were blended with Xelorex™ after Valley-Beater refining and made into DSF sheets.

FIG. 9 depicts a graph plotting MD (machine direction) tensile strength (in Kgf/mm) against Freeness (in CSF) for DSF (Dynamic Sheet Former) sheets made from the control pulp and the enzyme-treated pulp of FIG. 6. The pulps were blended with Xelorex™ after Valley-Beater refining and made into DSF sheets.

FIG. 10 depicts a graph plotting paper CD (cross-machine direction) tear strength (in gf) against Freeness (in CSF) for DSF (Dynamic Sheet Former) sheets made from the control pulp and the enzyme-treated pulp of FIG. 6. The pulps were blended with Xelorex™ after Valley-Beater refining and made into DSF sheets.

FIG. 11 depicts graph plotting Freeness (in CSF) against Valley-Beater refining (in minutes) for a control pulp and a pulp treated with a hyperthermostable cellulase, Cellulase B.

FIG. 12 depicts a graph plotting Freeness (in CSF) against Valley-Beater refining (in minutes) for a control pulp (no enzyme), pulp treated with Cellulase A at 55° C. and at three different dosages, and pulp treated with Cellulase B at 70° C. and at three different dosages. X=no enzyme-treated pulp. ▴=Cellulase B at 10 kg/T, 70° C. ▪=Cellulase B at 15 kg/T, 70° C. ●=Cellulase B at 20 kg/T, 70° C. White open symbols are data for Cellulase A at 55° C.; triangle dotted line is 0.2 kg/T data; square dotted line is 0.4 kg/T data; circle dotted line is 2 kg/T data.

FIG. 13 depicts a graph plotting length-weighted fiber length (LWAFL) against Valley-Beater refining (in minutes) for a control pulp (no enzyme), pulp treated with Cellulase A at 55° C. and at three different dosages, and pulp treated with Cellulase B at 70° C. and at two different dosages.

FIG. 14 depicts a graph plotting length-weighted fiber length (LWAFL) against Freeness (in CSF) for a control pulp (no enzyme), pulp treated with Cellulase A at 55° C. and at three different dosages, and pulp treated with Cellulase B at 70° C. and at two different dosages.

FIG. 15 depicts a graph plotting Freeness (in CSF) against Valley-Beater refining (in minutes) for a control pulp (no enzyme), control pulp co-refined with Percol®, and pulp treated with Cellulase A at 0.4 kg/T dosage and co-refined with either Percol® or Xelorex™.

FIG. 16 depicts a graph plotting length-weighted fiber length (LWAFL) against Freeness (in CSF) for a control pulp (no enzyme), control pulp co-refined with Percol, and pulp treated with Cellulase A at 0.4 kg/T dosage and co-refined with either Percol® or Xelorex™.

FIG. 17 depicts a graph plotting length-weighted fiber length (LWAFL) against Freeness (in CSF) for a control pulp (no enzyme), pulp treated with Cellulase A at 0.4 kg/T or 2 kg/T dosage, and pulp treated with Cellulase A at 0.4 kg/T, refined and then combined with either Percol® or Xelorex™.

FIG. 18 depicts a graph plotting Freeness (in CSF) against Valley-Beater refining (in minutes) for a control pulp (no enzyme) and pulp treated with Cellulase B, at 85° C.

FIG. 19 depicts a graph plotting Freeness (in CSF) against Specific Edge Load (SEL; in J/m) for a control pulp (no enzyme) and pulp treated with Cellulase A (as a dosage of 1 kg/T) at 50° C.

FIG. 20 depicts a graph plotting Freeness (in CSF) against Specific Refining Energy (KW-h/Ton) for a control pulp (no enzyme) and pulp treated with Cellulase A (as a dosage of 1 kg/T) at 50° C.

FIG. 21 depicts a graph plotting length-weighted fiber length (LWAFL) against Freeness (in CSF) for a control pulp for a control pulp (no enzyme), pulp treated with Cellulase A (as a dosage of 1 kg/T) at 50° C., with or without post-enzyme refining addition of Percol®.

FIG. 22 depicts a graph plotting length-weighted fiber length (LWAFL) Specific Refining Energy (KW-h/Ton) for a control pulp (no enzyme) and pulp treated with Cellulase A (as a dosage of 1 kg/T) at 50° C.

DESCRIPTION

There is a need in the art for fiber pulps having improved properties for papermaking and other products. The present disclosure addresses this need by provision of methods for preparing modified fiber pulps. Accordingly provided is a method of modifying papermaking fibers in a pulp to cause fiber interior damage to occur, wherein the modified pulp provides benefits in papermaking process, compared to non-modified pulps. The modified pulp provides at least one of the following benefits, related to corresponding non-modified pulps: (I) improved papermaking properties such as formation, which enables more softwood fiber to be used in place of hardwood fiber, (2) improved papermaking bulk at a given freeness; (3) improved papermaking smoothness; (4) reduced energy cost for refining; and (5) reduction in hemicellulose content. Additional benefits will be apparent based on the disclosure. The modified pulp and methods of its use are also provided.

Definitions

As used herein, each of the following terms has the meaning associated with it in this section. Additional definitions are present throughout the disclosure.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The term “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending on the context in which it is used. As used herein, the term “about” means that the number being described can deviate by plus or minus five percent of the number. For example, “about 250 g” means from 237.5-262.5 g. When the term “about” is used in a range, then the lower limit may be as much as minus 5% of the lower number and the upper limit may extend up to plus 5% of the upper number. For example, a range of about 100 to about 200 g indicates a range that extends from as low as 95 g up to 210 g.

Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

As envisioned in the present disclosure with respect to the disclosed compositions of matter and methods, in one aspect, the embodiments of the disclosure comprise the components and/or steps disclosed therein. In another aspect, the embodiments of the disclosure consist essentially of the components and/or steps disclosed therein. In yet another aspect, the embodiments of the disclosure consist of the components and/or steps disclosed therein.

DETAILED DESCRIPTION Fiber Length Reduction

In conventional papermaking, cellulase enzyme treatments are pursued either for drainage improvement or for refining strength improvement. In such cellulase enzyme treatments, interior fiber degradation, as reflected by fiber length reduction, is always avoided. Contrary to this conventional wisdom, Applicants have found that beneficial papermaking and paper product properties can be achieved by intentionally causing fiber morphology changes and fiber length reduction by a combination of cellulase treatment and refining as disclosed herein. More specifically, Applicants have found that inducing a controlled degree of fiber interior damage through the use of cellulolytic enzymes to produce a pulp having a reduction of papermaking fiber length of from 5% to 96% after going through the refiner, or by-passing the refiner, while maintaining a pulp freeness level of from 200 CSF to 700 CSF, relative to a non-treated pulp, possesses improved properties when used to produce paper, paper products, or other pulp products.

Provided herein is a method of modifying papermaking pulp fiber by contacting papermaking pulp with a cellulolytic enzyme under conditions to induce fiber interior damage, and with a fiber length reduction enhancement agent to produce a modified pulp having a reduction of papermaking fiber length of 5% to 96% after going through a refiner or after by-passing a refiner, and a pulp freeness level of from about 200 Canadian Standard Freeness (CSF) to about 700 CSF. The reduction of papermaking fiber length is with respect to a comparable pulp that is not subjected to the treatment with the cellulolytic enzyme and the fiber length reduction enhancement agent.

Exemplary dosages for the enzyme treatment are from 0.2 kilogram (of enzyme) per ton of pulp (kg/Ton) up to about 25 kg/Ton, from about 0.2 kilogram per ton of pulp (kg/Ton) up to about 20 kg/Ton, from about 2 kilogram per ton of pulp (kg/Ton) up to about 10 kg/Ton, or from about 0.2 kg/Ton up to about 2 kg/Ton.

The enzyme treatment can be carried out at temperature within the range of from about 40° to about 55° C., the typical paper mill temperature. Alternatively, the enzyme treatment can be carried out at a temperature greater than about 55° C., for instance, from about 70° C. to about 125° C. Exemplary cellulolytic enzymes for use in practicing the method in either temperature range are discussed in detail below. The cellulolytic enzyme, such as cellulase, can be used in combination with other enzymes commonly applied to the pulp and paper field, including hemicellulases, mannanases, xylanases, pectinases, esterases, lipases, manganese peroxidases, lignin peroxidases, laccases, dioxygenases, mono-oxygenases, chloroperoxidases, glyoxal oxidases, glucose oxidases, cellobiose dehydrogenases, and cellobiose quinone oxidoreductase.

The duration of the enzyme treatment should be sufficient to effect fiber interior damage, as reflected by fiber length reduction. For instance, the enzyme treatment duration can be 1 hour or long, or 1 to 10 hours.

The enzyme treatment can be carried out in any location from the post-digester pulp mill processing stages and up to the paper mill stock preparations, and wherein sufficient enzyme reaction time or temperature is provided. Exemplary locations for the enzyme treatment include refining stages, bleach plant stages, pulp high density (HD) towers, tanks, and pulpers/re-pulpers. In an embodiment, the enzyme treatment of the pulp fibers is carried out at the bleach plant stage, e.g., the bleached HD tower.

The enzyme contacting step can occur before or after Kraft pulping, before, during, or after oxygen delignification, before or after any bleaching, before or after any chemical pulping or modified chemical pulping, before or after semichemical pulping.

The fiber length reduction enhancement agent (also referred to herein as “length reduction enhancer”) comprises a highly hydrolyzed polymer or copolymer of vinylformamide with degree of hydrolysis >70%, a copolymer of acrylamide with acryloxyethyl-trimethylammonium chloride, a copolymer of acrylamide with diallyldimethylammonium chloride, a free glyoxal, or a mixture thereof. The length reduction enhancer is added prior to the enzyme treatment, during the enzyme treatment, or after the enzyme treatment.

The pulp freeness level of the modified pulp can be from about 200 CSF to about 700 CSF, from about 300 CSF to about 650 CSF, or from about 400 CSF to about 600 CF.

The modified pulp can be used in papermaking fiber furnish at 10% to 100% (blended with any untreated refined or untreated un-refined pulp sources), for the papermaking or forming of paper web materials.

Paper is commonly formed from wood fibers, namely softwoods and hardwoods. Main differences between softwood fibers and hardwood fibers are: the length of the individual cellulosic fibers of the wood, the coarseness of the fibers, and the stiffness or collapsibility of the fibers. Both smoothness and formation are affected by fiber length, morphology and collapsibility, among other things. It is well known that softwood fibers, although providing more strength than hardwood fibers, are substantially inferior to hardwood fiber in paper formation due to fiber length and printing smoothness due to fiber roughness/coarseness. Paper formation and printing smoothness are increasingly in high demand for packaging papers such as paper board and other high quality packaging and printing papers. This has become a strategically and economically important issue. For instance, in southeastern United States, where vast amount of pulp and paper products are manufactured, softwood fibers are more available than hardwood fibers.

Prior methods of modifying fiber morphology using conventional enzyme treatment (enzymatic refining) have been found inadequate and are not cost-effective. See, e.g., U.S. Pat. No. 8,262,850. Prior methods to address the drawbacks of enzymatic refining include strategies based on catalytic oxidations based on peroxides, as well as hypochlorite or hypochlorous acid strategies, in combination with refining. See, e.g., U.S. Pat. Nos. 8,262,850, 8,007,635, 8,282,774, and 8,753,484.

One advantage of the modified pulp produced by the disclosed method is modification of the fiber morphology of softwood fibers such that the modified softwood fibers can be used in place of hardwood fibers. The present disclosure provides strategies based on the combined contribution of enzymatic refining and select chemicals (fiber length reduction enhancement agents) for delivering a treated softwood fiber that is practical and cost-effective for the papermaking.

Accordingly, the method of the disclosure can be practiced with a pulp comprising or consisting of softwood fiber. In practicing the method of the disclosure, softwood fiber length can be reduced by 5% to 96% after going through the refiner, while maintaining pulp freeness level from about 200 CSF to about 700 CSF, from about 350 CSF to about 650 CSF, from about 350 CSF to about 650 CSF, or from about 400 CSF to about 650 CSF. The softwood fiber length can also be reduced by 10% to 96%, by 15% to 96%, or by 20% to 96%. Exemplary dosages for the enzyme treatment are from about 0.2 kilogram per ton of pulp (kg/Ton) up to about 25 kg/Ton, from about 0.2 kilogram per ton of pulp (kg/Ton) up to about 20 kg/Ton, from about 2 kilogram per ton of pulp (kg/Ton) up to about 10 kg/Ton, or from about 0.2 kg/Ton up to about 2 kg/Ton. The enzyme treatment can be carried out in any location from the post-digester pulp mill processing stages and up to the paper mill stock preparations, such bleach plant stages or pulp high density (HD) towers, and wherein sufficient enzyme reaction time or temperature is provided. Exemplary dosages of the fiber length reduction enhancement agent can be from 0.5 kg/ton to 20 kg/ton, or from 2 kg/Ton to 10 kg/Ton, prior to, during, or post the enzymatic refining stage.

The modified softwood fiber pulp can be used in papermaking fiber furnish at 10% to 100% (blended with any untreated refined or untreated un-refined pulp sources) for the papermaking or forming of paper web materials having improved papermaking properties such as formation. The improved properties advantageously allow more hardwood fibers to be replaced with the modified softwood fibers in papermaking processes.

In an aspect, the softwood fiber length is reduced by 10% to 96%, by 15% to 96%, or by 20% to 96%, with enzyme dosing from 0.2 kg/Ton to 2 kg/Ton, a fiber length reduction enhancement agent is a hydrolyzed polymer or copolymer of vinylformamide with degree of hydrolysis >70%, or a copolymer of acrylamide with acryloxyethyl-trimethylammonium chloride, and applied at a dosing from 0.5 kg/ton to 20 kg/ton, or from 2 kg/Ton to 10 kg/Ton (applied prior to, during, or after the enzyme treatment refining stage), and wherein pulp freeness can be from 300 CSF to 650 CSF, or from 400 CSF to 650 CSF.

In an aspect, the fiber length can be reduced by 5% to 20%, wherein the softwood or hardwood pulp is contacted with a length reduction enhancer agent before, during, or after the enzymatic refining treatment and pulp freeness of the modified pulp can be from 300 CSF and 650 CSF. The length reduction enhancer comprises a highly hydrolyzed polymer or copolymer of vinylformamide with degree of hydrolysis >70%, a copolymer of acrylamide with acryloxyethyl-trimethylammonium chloride, applied at dosages of from 0.5 kg/ton to 20 kg/ton, or from 2 kg/Ton to 10 kg/Ton, prior to, during, or post the enzymatic refining stage. Paper or paperboard products prepared using the modified softwood pulp are found advantageously to have improved papermaking bulk at given freeness.

In an aspect, the softwood fiber length can be reduced by 5% to 20%, wherein the softwood pulp is contacted with a length reduction enhancer agent before, during, or after the enzymatic refining treatment and pulp freeness of the modified pulp can be from 300 CSF and 650 CSF. Paper or paperboard products prepared using the modified softwood pulp is found advantageously to have improved papermaking bulk at given tensile strength.

In an aspect, the enzyme treatment can be performed in the presence of a fiber length reduction enhancement agent (“co-refining”), wherein softwood or hardwood fiber length is reduced by 5% to 20% and pulp freeness of the modified pulp can be from 300 CSF and 650 CSF. Paper or paperboard products prepared using the modified softwood pulp are found advantageously to have improved papermaking smoothness. The modified hardwood pulp can have reduced vessel picking/linting.

The enzyme treatment of the method as discussed above can also be done with any surfactants and/or dispersant, including anionic, nonionic, cationic, and zwitterionic surfactants and dispersants as has been well known to be used with cellulase enzyme hydrolysis and xylanase enzymes bleaching, as well as those surfactants and dispersants known for us in the pulping, washing, and papermaking systems.

The method of the disclosure can be practiced with a pulp comprising or consisting of hardwood fiber, to modify hardwood fiber morphology, increase fiber populations, and/or reduce coarseness of hardwood fiber, thereby upgrading hardwood fibers for tissues and personal care applications. For instance, some hardwood fibers can have high coarseness and fiber length, such as, but not limited to, US southern mixed hardwood pulps which can be modified to approach the fiber properties of more desirable fibers, such as Eucalyptus, Aspen, or Acacia. Further disclosure on fibers for practice of the disclosed methods is found in “Wood and Non-Wood Pulp Fibers.”

The method of the disclosure can also be used to treat pulp fibers for use in modifying dissolving pulps, and fluff pulps.

High Temperature Enzyme Treatment on Pulps

Modern mechanical pulping, such as thermo-mechanical pulping (TMP, CTMP, APMP), is done at very high temperatures, 70° C. to 120° C. at various pulping stage locations. This high temperature has prevented the use of cellulase enzymes in practical use for refining energy reductions, despite that various enzymes approaches including cellulases, hemicellulases, pectinases, laccases, and various peroxidases have been historically investigated for energy reductions at research stages. Lipase enzymes do not directly impact refining energy to any significant level, despite claiming to be helpful in removing pitch and extractives thus reducing some refining energy required for the fiber development.

Most commercially-available cellulases in the papermaking applications are enzymes which can only work effectively up to 50° C., or up to 55° C. Even if some cellulase enzymes may be extended to work at higher temperatures, these enzymes work much better or more effectively at lower temperatures.

Applicants have found that some cellulase enzymes that do not work at lower (i.e. typical papermaking) temperatures, work very effectively at temperatures substantially higher than papermaking processing temperatures.

Accordingly, this disclosure further provides a method of modifying pulp fiber comprising contacting pulp with a cellulolytic enzyme at a temperature from about 70° C. up to about 125° C., or from about 80° C. up to about 100° C. Preferably, the cellulolytic enzyme is substantially inactive at temperatures less than about 60° C. or less than about 55° C., such as from about 35° C. to about 55° C.

In one embodiment, the method advantageously yields a reduced energy expenditure for refining, for instance, a saving of refining electrical energy at either papermaking refining or at chip refining, of at least about 10%. The reduction of energy expenditure is with respect to a comparable pulp that is subjected to the same treatment at a temperature from about 70° C. up to about 125° C., but without enzymes. Example 8 illustrates such energy savings obtained with enzyme treatment at 85° C.

In another embodiment, the method can provide a modified pulp having improved papermaking properties. In an aspect, the hemicellulose content of the treated pulps after pulping and/or bleaching is reduced by at least about 0.2% wt. or more, when compared to pulps made without such enzyme treatment.

The high temperature range is above the operating temperature of paper mill sections (including stock preparation section, refiner section, paper machine section, and paper machine white water recirculations). The cellulase enzymes carried over by the treated papermaking fibers to the paper mill is preferably inactive at the typical paper mill temperature and thus would not affect other paper grades, and would not affect the chemistry of white water systems, thus enabling the long term paper machine operations. The modified pulps are therefore advantageous for use in numerous paper products. The high temperature method overcomes a big hurdle which has so far limited the broad use of cellulase enzymes in the paper mills.

Exemplary cellulolytic enzymes for use in practicing the method in a temperature range of about 70° C. up to about 125° C., from about 75° C. up to about 120° C., or from about 80° C. up to about 100° C. are discussed in detail below. Cellulolytic enzymes have enzymatic activity in these temperature ranges are referred to herein as thermostable and hyperthermostable enzymes. The thermostable and hyperthermostable cellulolytic enzyme can be substantially inactive at temperatures at later stages, such as at or below 60° C., or at or below 55° C., providing the additional advantage of not affecting other process streams or products in later papermaking stages.

Exemplary dosages for the high temperature enzyme treatment are from 0.2 kilogram (of enzyme) per ton of pulp (kg/Ton) up to about 25 kg/Ton, from about 0.2 kilogram per ton of pulp (kg/Ton) up to about 20 kg/Ton, from about 2 kilogram per ton of pulp (kg/Ton) up to about 10 kg/Ton, or from about 0.2 kg/Ton up to about 2 kg/Ton.

The cellulolytic enzyme treatment may optionally be done in combination with other hyperthermostable hemicellulases, such as hyperthermostable mannanase or hyperthermostable xylanases. For example, a few hyperthermostable xylanases between 70° C. and 90° C. and optionally up to 100° C., are described in WO 2007095398 (Weiner et al.), WO 2009045627 (Gray et al.), and WO2016073610 (Widner et al.).

The fibers can be wood or non-wood fibers or fibrous materials. The method of the disclosure can be practiced with a pulp comprising or consisting of hardwood fiber, to modify hardwood fiber morphology, increase fiber populations, and/or reduce coarseness of hardwood fiber, thereby upgrading hardwood fibers for tissues and personal care applications. Further disclosure on fibers for practice of the disclosed methods is found in “Wood and Non-Wood Pulp Fibers.” The fibers can be subject to treatment with mechanical defibrizing actions such as by an impressifiner, a grinder, a disc refiner, an extruder, a kneader, a homogenizer, a shredder, before, during, or after the hyperthermostable cellulolytic enzyme treatment step.

The duration of the enzyme treatment can be from about 30 minutes up to 10 hours.

The high temperature enzyme treatment can be carried out in any location from the post-digester pulp mill processing stages and up to the paper mill stock preparations, and wherein sufficient enzyme reaction time or temperature is provided. Exemplary locations for the enzyme treatment include refining stages, bleach plant stages, pulp high density (HD) towers, tanks, and pulpers/re-pulpers. The high temperature enzyme treatment can also be carried out as a pretreatment, for instance, at the impressifiner before refining, or can be done on refiner rejects before reject refining.

Optionally, any effective dispersant, penetrant, surfactants (anionic, nonionic, cationic, zwitterionic), and/or polymer carriers may be used in combination with the enzymes treatment.

As noted above, in an aspect of the high temperature enzyme treatment method, the hemicellulose content of the treated pulps is reduced. The method of modifying papermaking pulp fiber comprises contacting papermaking pulp with a cellulolytic enzyme, such as a cellulase enzyme, a hemicellulase enzyme comprising cellulase enzyme activities, or a mixture of cellulase and hemicellulase enzymes, at a temperature from about 70° C. up to about 125° C., or from about 80° C. up to about 100° C., in a reaction time of from about 30 minutes up to about 10 hours, wherein the modified pulp after pulping and/or bleaching has a hemicellulose content reduced by at least about 0.2% wt. or more, when compared to pulps made without high temperature enzyme treatment. Preferably, the cellulolytic enzyme is substantially inactive at temperatures less than about 60° C. or less than about 55° C., such as from about 35° C. to about 55° C.

The hyperthermostable enzyme can be applied before pulping, in-between pulping stages, and after pulping. The pulping is any mechanical pulping, any chemical pulping, any semichemical pulping, any semimechanical pulping, any chemi-mechanical pulping, recycling pulping, or deinking pulping. For instance the pulping can be sulfite pulping, Kraft pulping, prehydrolysis-Kraft Pulping, recycling pulping, solvent pulping, steam/hot water pretreatment (with or without explosion) pulping, ammonia (with or without explosion) pulping, sodium carbonate pulping, NSSC pulping, various TMP or RMP pulping, etc., as well as modified pulping disclosed, for instance in U.S. Pat. Nos. 8,328,983, 8,182,650, and 7,520,958. The hyperthermostable enzyme can also be applied before, during, or after bleach plant operations. The enzyme treatment step can also occur before, during, or after oxygen delignification.

The resulting modified pulps can be used as improved dissolving pulps with improved chemical reactivity for chemical derivatizations and reactions. The modified pulps may be used as a fluff pulps advantageously having one or more of reduced fluff shredding energies, modified liquid absorption capacities, modified surface free energy/contact angle, and modified liquid acquisition time. Such fluff pulps are useful for diapers or personal hygiene products. The modified pulps can be used as tissue pulps for softness in tissues. The modified pulp may be a papermaking pulp with increased bulk. Under certain conditions with such hyperthermostable cellulase treatment, the modified pulp can be further processed into microcrystalline cellulose, microfibrillated cellulose, nanofibrillated cellulose, nanocelluloses, and other specialty cellulose products. Biorefinery uses of the removed hemicellulose sugars, in lieu of modified pulps, is also contemplated, such as steam/hot water pretreatment (with or without explosion) pulping, ammonia (with or without explosion) pulping, sodium carbonate pulping, NSSC pulping, various TMP or RMP pulping, etc.

The high temperature treatment can be applied after Kraft pulping, such as in the Brown HD tower (with or without oxygen delignification upstream). The treatment can be applied after sulfite pulping. The treatment can also be applied before pulping. The treatment can be applied in-between the bleaching stages, and/or it can be applied after bleaching at the Bleached HD towers.

In an aspect, the high temperature treatment is used with wood or non-wood chips or fibrous materials, with a hyperthermostable cellulase and a hyperthermostable hemicellulase (e.g., xylanase, mannanase, and/or pectinase), preferably after mechanical defiberization, such as by an impressifiner or other mechanical device, followed by mechanical chemical (Kraft, sulfite, or solvent) or semi-chemical pulping. These constitute a modified pulping methods for paper, paper board, fluff pulps, tissue fibers, biorefinery (with sugar usages), or dissolving pulps. In addition to the hyperthermostable cellulase enzymes described previously, exemplary hyperthermostable xylanases include those described in patents WO 2007/095398 (Weiner et al.), WO 2009/045627 (Gray et al.), and WO2016/073610 (Widner et al.).

The paper mill temperature is usually between 40° C. and 55° C. Therefore, a cellulase enzyme that will not be active at these temperature ranges is preferred for practicing the disclosed method for papermaking fiber. Preferably the cellulase enzyme will be active only at greater than 55° C., or at greater than 60° C. In general, a cellulase enzyme that is only active from about 60° C. and 125° C., from about 65° C. and 125° C., or from about 70° C. and 125° C. can be used for delivering the fiber treatment and papermaking function in the disclosed method. This is the temperature range of hyperthermophiles.

In one aspect, the high temperature enzyme treatment is performed at the Bleached HD Tower (or a separate tank) in the end of bleach plant bleaching processes. Optionally, extra heating, such steam injection, or hot water feeding, may be applied. A reducing agent (such as SO₂, sulfite, or thiosulfate) can be optionally added to remove the residual oxidants (such as ClO₂ or peroxides), thus minimizing or preventing the inactivation of the cellulolytic enzymes by these bleach plant residual chemicals. For Bleached HD tower, the method is advantageously practiced with a cellulase enzyme that is only active at greater than 60° C., such as between 600 and 75° C.), or greater than 65° C., such as between 65° and 75° C.

If the enzyme temperature and pH tolerance is adequate, the high temperature enzyme treatment can be conducted before or in-between during the bleaching process, wherein after enzyme treatment, the bleaching process may serve to inactivate the enzymes, or the washer serve to remove the enzymes from treated fibers. For instance, the cellulase enzyme treatment can be carried out at a spare or redundant bleaching tower. A cellulolytic enzyme that is thermostable at greater than 65° C., or greater than 70° C., or greater than 75° C., can be compatible with the bleach plant stream process temperatures.

The high temperature enzyme treatment for papermaking pulp fiber can be carried out at the pulp mill, after chemical pulping or oxygen delignification, such as in the Brown HD Tank, where temperatures can be usually between 65° C. to 90° C., or 70° C. to 90° C. In the case of mechanical pulping, or chemi-mechanical pulping, the temperature can be between 70° C. to 120° C., depending on location in the pulp mill process.

Optionally, the enzyme treatment can be done in combination with an intracrystalline lignocellulosic agent. Exemplary intracrystalline lignocellulosic agents include ionic liquids, N-alkylated ureas, N-alkylated lactams, N-alkylated amides, polyol ethers, and polyols.

All the dispersants, penetrants, surfactants as disclosed previously, or known for use in the pulp and paper industry, can be included for use with the high temperature enzyme treatment or used as separate treatment.

Fiber Length Reduction—Softwood for Substitution of Hardwood Fibers

Provided herein is a method of modifying papermaking pulp softwood fiber by contacting the pulp having softwood fiber having a fiber length of about 2.1 mm to about 2.8 mm, as measured by FQA length-weighted fiber average length, with a cellulolytic enzyme at a temperature of about 35° C. to about 125° C., (2) refining the modified pulp, and thereby produce a modified pulp having (i) a fiber length of about 0.6 mm to about 1.7 mm, and (ii) a pulp freeness level of from about 400 Canadian Standard Freeness (CSF) to about 700 CSF. The reduction of papermaking fiber length is with respect to a comparable pulp that is not subjected to the treatment with the cellulolytic enzyme. Advantageously, the process permits a refining energy savings of at least 20% when compared to the control refining without enzyme treatment. In addition, the modified softwood fiber pulp advantageously may be substituted for hardwood fiber pulp in the manufacture of paper web or pulp products.

The enzyme treatment can be carried out in any location from the post-digester pulp mill processing stages and up to the paper mill stock preparations, and wherein sufficient enzyme reaction time or temperature is provided. Exemplary locations for the enzyme treatment include refining stages, bleach plant stages, pulp high density (HD) towers, tanks, and pulpers/re-pulpers. In an embodiment, the enzyme treatment of the pulp fibers is carried out at the bleach plant stage, e.g., the bleached HD tower.

The refining step is carried at sufficient refiner intensities with either a disc-refiner or conical refiner to obtain the appropriate freeness.

The contacting step can be carried out at a temperature of about 35° C. to about 125° C. for about 30 minutes to about 10 hours, or at a temperature of about 35° C. to about 125° for about 30 minutes to about 10 hours.

Exemplary dosages for the enzyme treatment are from 0.2 kilogram per ton of pulp (kg/Ton) up to about 25 kg/Ton, from about 0.2 kilogram per ton of pulp (kg/Ton) up to about 20 kg/Ton, from about 2 kilogram per ton of pulp (kg/Ton) up to about 10 kg/Ton, or from about 0.2 kg/Ton up to about 2 kg/Ton.

The enzyme treatment can be carried out at temperature within the range of from about 35° to about 65° C., the typical paper mill temperature. Alternatively, the enzyme treatment can be carried out at a temperature from about 65° C. to about 125° C. Exemplary cellulolytic enzymes for use in practicing the method in either temperature range are discussed in detail below. The cellulolytic enzyme, such as cellulase, can be used in combination with other enzymes commonly applied to the pulp and paper field, including hemicellulases, mannanases, xylanases, pectinases, esterases, lipases, manganese peroxidases, lignin peroxidases, laccases, dioxygenases, mono-oxygenases, chloroperoxidases, glyoxal oxidases, glucose oxidases, cellobiose dehydrogenases, and cellobiose quinone oxidoreductase.

Optionally, the pulp having softwood fiber is also contacted with a fiber length reduction enhancement agent. The pulp can be contacted with the fiber length reduction enhancement agent contact occurs either at the same time as contacting the pulp with the enzyme, or the pulp can be contacted after the enzyme contacting step and the refining step. The fiber length reduction enhancement agent comprises a highly hydrolyzed polymer or copolymer of vinylformamide with degree of hydrolysis >70%, a copolymer of acrylamide with acryloxyethyl-trimethylammonium chloride, a copolymer of acrylamide with diallyldimethylammonium chloride, a free glyoxal, a glyoxalated polyacrylamide, or mixtures thereof.

Recycling and Deinking with Hyperthermostable Cellulase

Thermostable and hyperthermostable cellulases enable enzymatic paper recycling and/or deinking at very high temperatures, such as greater than 70° C., greater than 75° C., or greater than 80° C. The recycling and/or deinking process can be practiced using thermostable or hyperthermostable cellulases from about 80° C. and about 125° C. The operation at very high temperatures enables the recycling to be more efficient in disintegrate strength or wet strength bonding, removing adhesives, softening and removing polymer additives, latexes. The high temperature process advantageously also allows softening and more efficient removal of ink toners, polymers, fats/fatty oils, and ink components.

In addition to the hyperthermostable cellulases and xylanases described above, recycling and/or deinking process can include hyperthermostable alpha-amylase enzymes, and other enzymes such as lipases and esterases. The hyperthermostable alpha amylase, include high temperature alpha-amylases, such as those commercially available from Novozymes and BASF Enzymes. An exemplary high temperature alpha-amylase is described in U.S. Pat. No. 7,273,740 (Callen et al.), which can be used at temperatures as high as 110° C., 120° C., and 125° C.

Thermostable and hyperthermostable enzymes may be used in combination with a deinking enhancing agent. Exemplary deinking enhancing agents include ionic liquids, N-alkylated ureas, N-alkylated lactams, N-alkylated amides, alkyl ethoxylates, phenyl ethoxylates, polyol ethers, and polyols. Exemplary deinking enhancing agents also include N-methyl pyrrolidone, N-ethyl pyrrolidone, cyclohexyl pyrrolidone, hexyl pyrrolidone, octyl pyrrolidone, Cellusolve (2-ethoxyethanol), and Butyl Cellusolve (2-Butoxyethanol). Exemplary ionic liquids include 1-ethyl-3-methyl imidazolium acetate, I-butyl-3-methyl imidazolium acetate, or other anion salts.

The deinking process may further include a mineral aid. Exemplary mineral aids include diatomaceous earth, calcined clays, hydrous clays, ground calcium carbonate, precipitated calcium carbonate, aluminum oxide, calcium silicates, chalk, and talc.

Cellulolytic Enzymes

Various cellulolytic enzymes can be used in the methods of the disclosure. Cellulolytic enzymes suitable for use in the disclosed methods can be active in a temperature range of from about 40° C. to 55° C., or can be thermostable or hyperthermostable cellulolytic enzymes, discussed further elsewhere herein.

Cellulolytic enzymes are primarily cellulase enzymes and/or cellulase-functioning hemicellulase enzymes. The cellulase enzymes include various types of endoglucanases, exoglucanases including various cellobiohydrolase types, and other frequently associated enzymes, such as glucosidases, etc. Pure endoglucanases, mixtures of cellulases, and complex of cellulolytic enzymes including cellulosomes, can be used in the disclosed methods. Cellulases isolated from, derived from, or modified from any origin, including bacterium, fungi, Archaea, animal, and plant origins, may be used in the disclosed method. Further, any isolated, synthetic or recombinant polypeptides having a cellulolytic activity are included. Additionally, a cellulase containing concomitant hemicellulase enzyme functions such as xylanases, mannanases, glucanases, and beta-glucanases may be used in the disclosed method.

The cellulases used in this disclosure may comprise any of the glycoside hydrolase GH families (Henrissat, 1991, Biochem. J. 280:309-316) containing cellulolytic functions, including GH families 1, 3, 5-9, 12, 26, 44, 45, 48, 51, 61, and 74. The cellulases may also be of any modular structures of catalytic domains, carbohydrate-binding domains, immunoglobulin domains, fibrinectin domains, or dockerin domains, etc. Various complexed and truncated cellulases can be used as well. Cellulases modified by any protein engineering tools, including various “directed evolution” strategies, and various rational designs can be used as well.

The cellulase enzyme used in the disclosed methods may contain various amount of beta-glucanase activities. Cellulase enzymes can also contain various types of hemicellulase enzyme activities. Included are also various hemicellulase enzymes or polypeptides containing concomitant cellulase functions, or displaying cellulases functions at certain environmental conditions such as pH, temperature, and ionic/salt ranges. A cellulase containing concomitant hemicellulase enzyme functions such as xylanases, mannanases, glucanases, and beta-glucanases may be used in the disclosed methods.

Exemplary cellulolytic enzymes useful in the disclosed methods include, but are not limited to, cellulases obtained or derived from Chrysosporium lucknowense/Myceliophthora thermophilia, cellulases obtained or derived from Humicola insolens (or produced from Aspergillus), and cellulases obtained or derived from Trichoderma. Exemplary cellulolytic enzymes useful in the disclosed methods are commercially available. A few exemplary commercially available cellulases are cited, but are not intended as exhaustive listing at all. Exemplary enzymes from Novozymes (Denmark) include Novozym® 613, Novozym® 476, and products in the Celluzyme®, Celluclast®, Carezyme®, and FiberCare® families. FibreZyme® G4 powder or liquid is an exemplary enzyme from Dyadic International (Jupiter, Fla.). Exemplary enzymes from Genencor (DuPont) include Multifect® A40, and cellulases from the Pergalase® or Optimase® families. ONOZUKA cellulases (Yakult Pharmaceutical Industry Co, Ltd, Japan) are further exemplary enzymes. Pyrolase® and Pyrolase®200, thermostable cellulases, and Pyrolase®HT, a hyperthermostable cellulase, from BASF Enzymes LLC (San Diego, Calif.) are further exemplary enzymes for the disclosed methods. Some commercially formulated biomass/biorefinery hydrolysis cellulases, such as Cellic® CTec, CTec2, and CTec3 from Novozymes; Accellerase™ 1000, Accellerase™ 1500, and Accellerase™ TRIO from DuPont; and AlternaFuel CMAX from Dyadic, can also be used in the disclosed methods. One particular cellulase enzyme product is Biocellulase W (from Kerry Food Ingredients Ltd.), which is made from Tricoderma reesei.

Cellulases can be produced from vast sources of microorganisms. They can be commonly produced from brown rot fungi, white rot fungi, soft rot fungi, bacteria, and actinomycetes. To name a few of the genera, they include Aspergillus (such as A. niger, A. oryzae, etc.), Melanocarpus (M. albomyces), Humicola (such as H. insolens and H. grisea), Tricoderma (such as T. reesei, T. viride, and T. longibrachiatum), Fusarium (such as F. oxysporium), Myceliophthora, Penicillium, Bacillus (such as B. subtilis and B. licheniformis), Pseudomonas (e.g., P. cellulosa); Rhodothermus, Phanerochaete, Trametes, Clostridium (e.g., C. thermocellum), Cellulomonas (e.g., C. fimi), Streptomyces, and Thermonospora (e.g., T. fusca). Some of the microbial organisms containing cellulase activities may reside in the rumens of cattles or other ruminating mammals, while cellulase activities from the symbiotic microorganisms are also found in the guts of some arthropods such as termites, and possibly even certain grass hoppers, or cockroaches.

A few of the commercial cellulase producing origins may be cited here as examples, without being limited by them. For instance, Chrysosporium lucknowense/Myceliophthora thermophilia (the “C1 Technology”) is used by DuPont (formerly Dyadic) for the G4 cellulase enzyme productions. All the cellulase enzymes, or cellulase-functioning polypeptides isolated, synthetic, or recombinants from the DuPont C1 Technology, including all variations, modifications, and formulations, are suitable as the enzymes for the disclosed methods. Exemplary cellulases are also disclosed in U.S. Pat. Nos. 7,892,812, 7,883,872, 8,673,618, and 8,916,363. As another example, Humicola insolens and related molecular biology modifications are used as origins by Novozymes for cellulase production. Melanocarpus albomyces is used for AB Enzymes cellulase. As a source of industrial cellulase enzymes, Trichoderma is also frequently used. For instance, Tricoderma reesei is known to produce significant exo- and endo-cellulase enzyme activities, together with significant beta-glucanases, xylanases, mannanases, and hemicellulases in general. All of these Tricoderma derived enzymes and enzyme functions, either through natural fermentations or through various genetic modifications, including various enzyme modules, domains, and fusion-proteins, are all included. The cellulase enzymes with broad beta glucanase and/or hemicellulase activities, sometimes, may provide synergistic actions on lignocellulosic fibers including OCC pulps, Kraft pulps, mechanical pulps, semi-chem pulps, paper pulps, fluff pulps and dissolving pulps. For fluff pulps, the enzyme would modify the surface fiber chemistry and hemicellulose distributions as well as fiber smoothness/surface morphology, which may be helpful for liquid transport or wicking, while minimizing the fiber length reductions. For dissolving pulps, the cellulase would reduce pulp viscosity (as required by dissolving pulp specifications, usually below 10 cps, or below 7 cps.) while further removing xylan, glucomannan, and other hemicellulose content. In one embodiment, the enzymes or polypeptides comprising both cellulase and hemicellulase activities are fused proteins. It should be noted that, despite the noted microbial origins, the final commercial production of cellulases may be made from a different host organism. Non-limiting examples of host production organisms include Aspergillus, Bacillus, E. coli, Pseudomonas, Saccharomyces, and Pichia pastoris. Various molecular biology and protein engineering tools may be applied, before the enzymes are produced industrially. All these cellulase enzymes, polypeptides containing cellulase activities, including all their variants, modifications, provided the appropriate enzymatic activity is retained, are suitable for use in the disclosed methods.

Thermostable and Hyperthermostable Enzymes

The thermostable and hyperthermostable cellulase enzymes useful in the disclosed methods are active at temperatures greater than 60° C., greater than 65° C., or greater than 70° C. These temperatures exceed the papermaking temperature range of 40° C. to 55° C.

Particular examples of the thermostable and hyperthermostable cellulase enzymes include cellulase enzyme products from BASF Enzymes. As demonstrated herein, treatment with cellulase of SEQ ID NO: 6 on fibers, even at the upper range of papermaking (paper machine) temperature of 55° C., did not produce the fiber length reduction after post-refining of the disclosed method. However, this cellulase worked very effectively in fiber length reduction at or above 70° C. This inaction of the cellulase of SEQ ID NO: 6 at papermaking temperatures followed by refining, is an unexpected finding. For the present disclosure, a temperature range of 60° C. to 95° C., preferably 65° C. to 90° C., more preferably 70° C. to 85°C. is recommended for cellulase of SEQ ID NO: -6. SEQ ID NOS: 5 and 7 are exemplary coding sequences for the cellulase of SEQ ID NO: 6. Higher temperatures are recommended for some other kind of cellulases. As an illustration, for the cellulase of SEQ ID NO: 2, the recommended temperature range for enzyme treatment is between 65° C. to 120° C., preferably 70° C. to 120° C., or more preferably between 80° C. to 115° C. The pH range can also vary from pH 4 to pH 12, pH 5 to pH12, pH 6 to pH12, pH 6 to pH 11, pH 6 to pH 10.5, pH 6 to pH 10, or pH 6 to pH 9.5. Exemplary coding sequences for the cellulase of SEQ ID NO: 2 include SEQ ID NO: 1 and SEQ ID NO: 3.

Furthermore, cellulases, such as exemplary cellulases of SEQ ID NOs: 2 and 6, can be processed to further improve activity. For instance, they can be concentrated (such as by membranes) to higher enzyme concentrations (thus activities) during manufacturing. They can also be dried, such as by lyophilization or by spray drying, with inert and/or protective materials to powders with higher enzyme content (thus activities) during enzyme manufacturing. The inert materials can be various types of clays, kaolin, bentonites, mineral particles, aluminum oxides, calcium silicates, calcium carbonates, talc, silica gels, silica oxides, sands, diatomaceous earth, zeolites, sorbitol, xylitol, mannitol, maltodextrins, trehalose, resistance starch, etc. The protective materials, if used, may include glycerol, low molecular PEGs, medium to high molecular weight PEGs, Tweens, polyethylene oxide, polyacrylamides, anionic polyacrylamides, glycerol esters such as ethoxylates, PEG esters such as ethoxylates, and phenol esters such as ethoxylates, albumins, proteins, soy proteins, milk proteins, casein, sodium caseinate, whey proteins, milk protein isolates, gelatins, low viscosity alginates, low viscosity CMC, low viscosity gellan gums, low viscosity polyvinylpyrrolidone (PVP).

Cellulases can also be further improved on fermentation yields by both molecular biology as well as process improvements.

In a broad sense, hyperthermostable enzymes can be derived from hyperthermophilic microorganisms. They can also be further improved, modified, synthesized, or recombined through any known molecular biology and protein engineering tools beyond the wild types origins. These tools include both random mutations and site-specific mutations.

Preferably these enzymes are derived from hyperthermostable microorganisms. Examples of hyperthermophiles include bacteria, such as Thermotoga (e.g., various Thermotoga maritima strains, T. neapolitana, and T. thermarum), Thermocrinis (T. ruber), and Aquifex (A. pyrophilus, and A. aeolicus). Some thermophile bacteria may be related to hyperthermophile, such as Calderobacterium hydrogenophilum, Thermosipho africanus, and Fervidobacterium hydrogenophilum. Cellulase enzyme from bacterium, Rhodothermus marinus, has also been reported to behave as hyperthermostable cellulase that was stable at 90° C. and 100° C. (Hreggvidsson et al. (1996) Appl. Env. Microbiology, 62(8): 3047-3049). Extreme thermostable examples also include enzymes isolated or genetically engineered from various strains of Dictyoglomus thermophilum.

Examples of hyperthermophile Archaea and some related Archaea, include: Sulfolobus (S. acidocaldarius, S. solfatarius), Metallosphaera (M. sedula), Acidianus (A. infernus), Stygiolobus azoricus, Sulfurococcus mirabilis, Sulfurisphaera ohwakuensis, Thermoproleus tenax, Pyrobaculum (P. islandicum, P. aerophilum), Thermofilum (T. pendens, T. librum), Thermocladium (T. modestius), Caldivirga (C. maquilingensis), Desulfurcoccus (D. mucosus, D. fermentans), Slaphylothermus (S. marinus), Sulfophohococcus (S. zilligii), Stetleria (S. hydrogenophila), Aeropyrum (A. pernix), Ignicoccus (I. islundicus), Thermosphaera (T. aggregans), Thermodiscus (T. maritimus), Pvrodiclium (P. occultum), Hyperthermus (H. butylicus), Pyrolobus (P. fumarii), Thermnococcus (T. cele), Pyrococcus (P. furiosus, P. abyssi. P. horikoshii, P. woesii), Archcaeoglobus (A. fulgidus, A. veneficus, A. profundus), Ferroglobus (F. placidus), Methanoihermus (M. fervidus, M. sociabilis), Methanococcus (M. janaschii, M. igneus, M. thermolitho-trophicus), Methanopyrus (M. kandleri), and Thermoplasma (T. acidophilum, T. volcanium).

All the cellulases originated from the above hyperthermophilic bacteria and hyperthermophilic Archaea, as well as the related bacteria and Archaea, can be used in the disclosed methods. Furthermore, any cellulases and beta glucanases encoded by genes or containing segments of such genes derived, isolated, modified, or synthesized from the above hyperthermophiles, are included in the present disclosure, provided they exhibit the appropriate enzymatic activity. Cellulases improved by directed evolution, by site-specific mutations, or other approaches such as cellulase enhancement through enrichment of a consortium of hyperthermophiles growing on the proper cellulose substrates (Graham et al. (2011) Nature Communications), can be used in the disclosed methods.

Particularly mentioned for use in the disclosed methods are cellulases (endoglucanases, exoglucanases) derived from Thermotoga sp. strain FjSS3-B.1, Thermotoga maritima MSB8, and Thermotoga neapolitana (Sunna et al. (1997) “Glycosyl hydrolases from hyperthermophiles,” Extremophiles 1: 2-13). All the cellulases from Thermotoga maritima as described in U.S. Pat. Nos. 5,925,749, 5,962,258, 6,008,032, and 6,245,547 are suitable for use in the disclosed methods. Also useful are the endoglucanases described by U.S. Pat. Nos. 5,789,228, 6,001,984, 6,074,867, 6,329,187, and 7,465,571, particularly the endoglucanases from “AEPII 1a.” All the cellulases or glucanases, and their polypeptide sequences and fragments as taught in U.S. Pat. No. 7,960,148 (Steer et al. 2011), US Publication No. 20140295523 (Steer et al. 2014), U.S. Pat. No. 7,422,876 (Short et al.) and U.S. Pat. No. 8,426,184 (Blum et al.) are suitable for use in the disclosed methods. Modifications and variations of various nucleotide sequences that encoding cellulases or polypeptides in various combinations, such as in US Publication No. 20150072397, including mutations related to Thermotoga maritima cellulases, are suitable for the cellulases for the disclosed methods. Methods used for enzymatic modifications, include error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR), Chromosomal Saturation Mutagenesis (CSM), or a combination thereof. In another aspect, the modification methods also include recombination, recursive sequence recombination, phosphothiolate-modified NDA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purifications mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and combination thereof.

Some cellulases derived from hyperthermophilic bacteria and/or non-naturally occurring variants thereof are described in PCT publication WO 2009/020459; the entire disclosure of which is incorporated herein by reference thereto. Included within the entire specification of the WO 2009/020459 publication, the entirety of which is hereby incorporated by reference are the below-listed DNA and amino acid SEQ ID NOS. These include:

-   -   WO 2009/020459 SEQ ID NOS: 1 and 2 (wild-type ‘parent’ T.         maritima cellulase), disclosed herein as SEQ ID NOS: 5 and 6,         respectively;     -   WO 2009/020459 SEQ ID NO: 3 (wild-type DNA, altered to remove         alternate starts) disclosed herein as SEQ ID NO: 7;     -   WO 2009/020459 SEQ ID NOS: 6 and 7 (“7X” combined Gene Site         Saturation Mutagenesis (“GSSM”) mutations) disclosed herein as         SEQ ID NOS: 8 and 9, respectively;     -   WO 2009/020459 SEQ ID NOS: 8 and 9 (“12X-6” combined GSSM         mutations), disclosed herein as SEQ ID NOS: 3 and 2,         respectively;     -   WO 2009/020459 SEQ ID NOS: 10 and 11 (“13X-1” combined GSSM         mutations) disclosed herein as SEQ ID NOS: 10 and 11,         respectively;     -   WO 2009/020459 SEQ ID NOS: 12, and 13 (“12X-1” combined GSSM         mutations) disclosed herein as SEQ ID NOS: 12 and 13,         respectively;     -   WO 2009/020459 SEQ ID NOS: 16 and 17 (alternative cellulase         breaker from Thermotoga sp.) disclosed herein as SEQ ID NOS: 14         and 15, respectively;     -   WO 2009/020459 SEQ ID NOS: 18 and 19 (“7X” codon-optimized         version of T. maritima cellulase for maize expression) disclosed         herein as SEQ ID NOS: 16 and 17, respectively; and     -   WO 2009/020459 SEQ ID NOS: 20 and 21 (“12X-6” codon-optimized         version of T. maritima cellulase for maize expression) disclosed         herein as SEQ ID NOS: 18 and 19, respectively.

Besides the above-listed nucleotide and amino acid sequences related to wild-type and evolved variants of the cellulase from Thermotoga maritima strain MSB8, the additional mutants listed in Table 2 and Example 5 from WO 2009/020459, are also useful cellulase enzymes in the practice of the disclosed methods. The cellulases encoded by SEQ ID NOS: 1, 4, and 20 and the cellulase of SEQ ID NO: 21 are other cellulases useful in the disclosed methods.

In addition to the hyperthermostable cellulase enzymes, exemplary hyperthermostable xylanases include those described in patents WO 2007/095398 (Weiner et al.), WO 2009/045627 (Gray et al.), and WO2016/073610 (Widner et al.). SEQ ID NO: 22 is the sequence of a xylanase useful in the disclosed methods. SEQ ID NO: 23 is an exemplary coding sequence for the xylanase of SEQ ID NO: 22.

Sequences of Thermostable and Hyperthermostable Cellulases and Hemicellulases

Embodiments disclosed herein may use hyperthermostable polypeptides having cellulases activities or in combinations with hemicellulase activities, at temperatures from 70° C. to 125° C., optionally from 80° C. to 100° C., for their uses in pulp and paper production. In some embodiments, the hyperthermostable cellulase and/or hemicellulases are from bacteria, fungi, or Archaea, or any combination (mixture) thereof. Further useful in the disclosed methods can be isolated, synthetic or recombinant polypeptides having cellulase activity, (i) comprising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or has 100% (complete) sequence identity to any one of the exemplary polypeptide as described herein, wherein in one aspect (optionally) the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.

Table 1 lists exemplary thermostable and hyperthermostable cellulases and hemicellulases that can be used in the methods of the disclosure.

TABLE 1 SEQ ID NO Sequence  1 atgggcgtcgatccgtttgaacgtaacaaaatcttgggccgcggcattaatatcggcaatgcgctcgaagcaccaa atgaaggcgactggggagtggtgataaaagatgagttcttcgacattataaaagaagccggtttctctcatgttcg aattccaataagatggagtacgcacgctcaggcgtttcctccttataaaatcgagccttctttcttcaaaagagtg gatgaagtgataaacggagccctgaaaagaggactggctgttgttataaatattcatcactacgaggagttaatga atgatccagaagaacacaaggaaagatttcttgctctttggaaacaaattgctgatcgttataaagactatcccga aactctattttttgaaattctgaatgaacctcacggaaatcttactccggaaaaatggaatgaactgcttgaggaa gctctaaaagttataagatcaattgacaaaaagcacactgtgattataggcacagctgaatgggggggtatatctg cccttgaaaaactgagggtcccaaaatgggaaaaaaatgcgatagttacaattcactactacaatcctttcgaatt tacccatcaaggagctgagtgggtgcctggatctgagaaatggttgggaagaaagtggggatctccagatgatcag aaacatttgatagaagaattcaattttatagaagaatggtcaaaaaagaacaaaagaccaatttacataggtgagt ttggtgcctacagaaaagctgaccttgaatcaagaataaaatggacctcctttgtcgttcgcgaagccgagaaaag ggggtggagctgggcatactgggaattttgttccggttttggtgtttatgatcctctgagaaaacagtggaataaa gatcttttagaagctttaataggaggagatagcattgaatga  2 MGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTHAQAFPPYKIEPSFFKRV DEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIADRYKDYPETLFFEILNEPHGNLTPEKWNELLEE ALKVIRSIDKKHTVIIGTAEWGGISSLEKLRVPKWEKNAIVTIHYYNPFEFTHQGAEWVPGSEKWLGRKWGSPDDQ KHLIEEFNFIEEWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRGWSWAYWEFCSGFGVYDPLRKQWNK DLLEALIGGDSIE  3 atgggtgttgatccttttgaaaggaacaaaatattgggaagaggcattaatataggaaatgcgcttgaagcaccaa atgagggagactggggagtggtgataaaagatgagttcttcgacattataaaagaagccggtttctctcatgttcg aattccaataagatggagtacgcacgctcaggcgtttcctccttataaaatcgagccttctttcttcaaaagagtg gatgaagtgataaacggagccctgaaaagaggactggctgttgttataaatattcatcactacgaggagttaatga atgatccagaagaacacaaggaaagatttcttgctctttggaaacaaattgctgatcgttataaagactatcccga aactctattttttgaaattctgaatgaacctcacggaaatcttactccggaaaaatggaatgaactgcttgaggaa gctctaaaagttataagatcaattgacaaaaagcacactgtgattataggcacagctgaatgggggggtatatctg cccttgaaaaactgagggtcccaaaatgggaaaaaaatgcgatagttacaattcactactacaatcctttcgaatt tacccatcaaggagctgagtgggtgcctggatctgagaaatggttgggaagaaagtggggatctccagatgatcag aaacatttgatagaagaattcaattttatagaagaatggtcaaaaaagaacaaaagaccaatttacataggtgagt ttggtgcctacagaaaagctgaccttgaatcaagaataaaatggacctcctttgtcgttcgcgaagccgagaaaag ggggtggagctgggcatactgggaattttgttccggttttggtgtttatgatcctctgagaaaacagtggaataaa gatcttttagaagctttaataggaggagatagcattgaataa  4 tctactagttaggaggtaacttatgggcgtcgatccgtttgaacgtaacaaaatcttgggccgcggcattaatatc ggcaatgcgctcgaagcaccaaatgaaggcgactggggagtggtgataaaagatgagttcttcgacattataaaag aagccggtttctctcatgttcgaattccaataagatggagtacgcacgctcaggcgtttcctccttataaaatcga gccttctttcttcaaaagagtggatgaagtgataaacggagccctgaaaagaggactggctgttgttataaatatt catcactacgaggagttaatgaatgatccagaagaacacaaggaaagatttcttgctctttggaaacaaattgctg atcgttataaagactatcccgaaactctattttttgaaattctgaatgaacctcacggaaatcttactccggaaaa atggaatgaactgcttgaggaagctctaaaagttataagatcaattgacaaaaagcacactgtgattataggcaca gctgaatgggggggtatatctgcccttgaaaaactgagggtcccaaaatgggaaaaaaatgcgatagttacaattc actactacaatcctttcgaatttacccatcaaggagctgagtgggtgcctggatctgagaaatggttgggaagaaa gtggggatctccagatgatcagaaacatttgatagaagaattcaattttatagaagaatggtcaaaaaagaacaaa agaccaatttacataggtgagtttggtgcctacagaaaagctgaccttgaatcaagaataaaatggacctcctttg tcgttcgcgaagccgagaaaagggggtggagctgggcatactgggaattttgttccggttttggtgtttatgatcc tctgagaaaacagtggaataaagatcttttagaagctttaataggaggagatagcattgaatga  5 atgggtgttgatccttttgaaaggaacaaaatattgggaagaggcattaatataggaaatgcgcttgaagcaccaa atgagggagactggggagtggtgataaaagatgagttcttcgacattataaaagaagccggtttctctcatgttcg aattccaataagatggagtacgcacgcttacgcgtttcctccttataaaatcatggatcgcttcttcaaaagagtg gatgaagtgataaacggagccctgaaaagaggactggctgttgttataaatattcatcactacgaggagttaatga atgatccagaagaacacaaggaaagatttcttgctctttggaaacaaattgctgatcgttataaagactatcccga aactctattttttgaaattctgaatgaacctcacggaaatcttactccggaaaaatggaatgaactgcttgaggaa gctctaaaagttataagatcaattgacaaaaagcacactataattataggcacagctgaatgggggggtatatctg cccttgaaaaactgtctgtcccaaaatgggaaaaaaattctatagttacaattcactactacaatcctttcgaatt tacccatcaaggagctgagtgggtggaaggatctgagaaatggttgggaagaaagtggggatctccagatgatcag aaacatttgatagaagaattcaattttatagaagaatggtcaaaaaagaacaaaagaccaatttacataggtgagt ttggtgcctacagaaaagctgaccttgaatcaagaataaaatggacctcctttgtcgttcgcgaaatggagaaaag gagatggagctgggcatactgggaattttgttccggttttggtgtttatgatactctgagaaaaacctggaataaa gatcttttagaagctttaataggaggagatagcattgaataa  6 MGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTHAYAFPPYKIMDRFFKRV DEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIADRYKDYPETLFFEILNEPHGNLTPEKWNELLEE ALKVIRSIDKKHTIIIGTAEWGGISALEKLSVPKWEKNSIVTIHYYNPFEFTHQGAEWVEGSEKWLGRKWGSPDDQ KHLIEEFNFIEEWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREMEKRRWSWAYWEFCSGFGVYDTLRKTWNK DLLEALIGGDSIE  7 atgggtgttgatccttttgaaaggaacaaaatattgggaagaggcattaatataggaaatgcgcttgaagcaccaa atgagggcgactggggagtcgtgataaaagatgagttcttcgacattataaaagaagccggtttctctcatgttcg aattccaataagatggagtacgcacgcttacgcgtttcctccttataaaatcatggatcgcttcttcaaaagagtg gatgaagtgataaacggagccctgaaaagaggactggctgttgttataaatattcatcactacgaggagttaatga atgatccagaagaacacaaggaaagatttcttgctctttggaaacaaattgctgatcgttataaagactatcccga aactctattttttgaaattctgaatgaacctcacggaaatcttactccggaaaaatggaatgaactgcttgaggaa gctctaaaagttataagatcaattgacaaaaagcacactataattataggcacagctgaatgggggggtatatctg cccttgaaaaactgtctgtcccaaaatgggaaaaaaattctatagttacaattcactactacaatcctttcgaatt tacccatcaaggagctgagtgggtggaaggatctgagaaatggttgggaagaaagtggggatctccagatgatcag aaacatttgatagaagaattcaattttatagaagaatggtcaaaaaagaacaaaagaccaatttacataggtgagt ttggtgcctacagaaaagctgaccttgaatcaagaataaaatggacctcctttgtcgttcgcgaaatggagaaaag gagatggagctgggcatactgggaattttgttccggttttggtgtttatgatactctgagaaaaacctggaataaa gatcttttagaagctttaataggaggagatagcattgaataa  8 atgggtgttgatccttttgaaaggaacaaaatattgggaagaggcattaatataggaaatgcgcttgaagcaccaa atgagggagactggggagtggtgataaaagatgagtatttcgacaattataaaagaagccggtttctctcatgttc gaattccaataagatggagtacgcacgctcaggcgtttcctccttataaaatcgaggatcgcttcttcaaaagagt ggatgaagtgataaacggagccctgaaaagaggactggctgttgttataaatcagcatcactacgaggagttaatg aatgatccagaagaacacaaggaaagatttcttgctctttggaaacaaattgctgatcgttataaagactatcccg aaactctattttttgaaattctgaatgaacctcacggaaatcttactccggaaaaatggaatgaactgcttgagga agctctaaaagttataagatcaattgacaaaaagcacactataattataggcacagctgaatgggggggtatatct gcccttgaaaaactgagggtcccaaaatgggaaaaaaatgcgatagttacaattcactactacaatcctttcgaat ttacccatcaaggagctgagtgggtggaaggatctgagaaatggttgggaagaaagtggggatctccagatgatca gaaacatttgatagaagaattcaattttatagaagaatggtcaaaaaagaacaaaagaccaatttacataggtgag tttggtgcctacagaaaagctgaccttgaatcaagaataaaatggacctcctttgtcgttcgcgaagctgagaaaa ggagatggagctgggcatactgggaattttgttccggttttggtgtttatgatactctgagaaaaacctggaataa agatcttttagaagctttaataggaggagatagcattgaataacaccattccaagatggcgtg  9 MGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEYFDIIKEAGFSHVRIPIRWSTHAQAFPPYKIEDRFFKRV DEVINGALKRGLAVVINQHHYEELMNDPEEHKERFLALWKQIADRYKDYPETLFFEILNEPHGNLTPEKWNELLEE ALKVIRSIDKKHTIIIGTAEWGGISALEKLRVPKWEKNAIVTIHYYNPFEFTHQGGAEWVEGSEKWLGRKWGSPDD QKHLIEEFNFIEEWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRRWSWAYWEFCSGFGVYDTLRKTWN KDLLEALIGGDSIEHHS 10 atgggtgttgatccttttgaaaggaacaaaatattgggaagaggcattaatataggaaatgcgcttgaagcaccaa atgagggagactggggagtggtgataaaagatgagtatttcgacattataaaagaagccggtttctctcatgttcg aattccaataagatggagtacgcacgctcaggcgtttcctccttataaaatcgaggattctttcttcaaaagagtg gatgaagtgataaacggagccctgaaaagaggactggctgttgttataaatattcatcactacgaggagttaatga atgatccagaagaacacaaggaaagatttcttgctctttggaaacaaattgctgatcgttataaagactatcccga aactctattttttgaaattctgaatgaacctcacggaaatcttactccggaaaaatggaatgaactgcttgaggaa gctctaaaagttataagatcaattgacaaaaagcacactgtgattataggcacagctgaatgggggggtatatctg cccttgaaaaactgagggtcccaaaatgggaaaaaaatgcgatagttacaattcactactacaatcctttcgaatt tacccatcaaggagctgagtgggtgcctggatctgagaaatggttgggaagaaagtggggatctccagatgatcag aaacatgtgatagaagaattcaattttatagaagaatggtcaaaaaagaacaaaagaccaatttacataggtgagt ttggtgcctacagaaaagctgaccttgaatcaagaataaaatggacctcctttgtcgttcgcgaagccgagaaaag ggggtggagctgggcatactgggaattttgttccggttttggtgtttatgatcctctgagaaaacagtggaataaa gatcttttagaagctctaataggaggagatagcattgaataa 11 MGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEYFDIIKEAGFSHVRIPIRWSTHAQAFPPYKIEDSFFKRV DEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIADRYKDYPETLFFEILNEPHGNLTPEKWNELLEE ALKVIRSIDKKHTVIIGTAEWGGISALEKLRVPKWEKNAIVTIHYYNPFEFTHQGAEWVPGSEKWLGRKWGSPDDQ KHVIEEFNFIEEWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRGWSWAYWEFCSGFGVYDPLRKQWNK DLLEALIGGDSIE 12 atgggtgttgatccttttgaaaggaacaaaatattgggaagaggcattaatataggaaatgcgcttgaagcaccaa atgagggagactggggagtggtgataaaagatgagttcttcgacattataaaagaagccggtttctctcatgttcg aattccaataagatggagtacgcacgctcaggcgtttcctccttataaaatcgaggattctttcttcaaaagagtg gatgaagtgataaacggagccctgaaaagaggactggctgttgttataaatcagcatcactacgaggagttaatga atgatccagaagaacacaaggaaagatttcttgctctttggaaacaaattgctgatcgttataaagactatcccga aactctattttttgaaattctgaatgaacctcacggaaatcttactccggaaaaatggaatgaactgcttgaggaa gctctaaaagttataagatcaattgacaaaaagcacactgtgattataggcacagctgaatgggggggtatatctg cccttgaaaaactgagggtcccaaaatgggaaaaaaatgcgatagttacaattcactactacaatcctttcgaatt tacccatcaaggagctgagtgggtgcctggatctgagaaatggttgggaagaaagtggggatctccagatgatcag aaacatttgatagaagaattcaattttatagaagaatggtcaaaaaagaacaaaagaccaatttacataggtgagt ttggtgcctacagaaaagctgaccttgaatcaagaataaaatggacctcctttgtcgttcgcgaagccgagaaaag ggggtggagctgggcatactgggaattttgttccggttttggtgtttatgatcctctgagaaaacagtggaataaa gatcttttagaagctttaataggaggagatagcattgaataa 13 MGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTHAQAFPPYKIEDSFFKRV DEVINGALKRGLAVVINQHHYEELMNDPEEHKERFLALWKQIADRYKDYPETLFFEILNEPHGNLTPEKWNELLEE ALKVIRSIDKKHTVIIGTAEWGGISALEKLRVPKWEKNAIVTIHYYNPFEFTHQGAEWVPGSEKWLGRKWGSPDDQ KHLIEEFNFIEEWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRGWSWAYWEFCSGFGVYDPLRKQWNK DLLEALIGGDSIE 14 atggaacagtcagttgctgaaagtgatagcaactcagcatttgaatacaacaaaatggtaggtaaaggagtaaata ttggaaatgctttagaagctcctttcgaaggagcttggggagtaagaattgaggatgaatattttgagataataaa gaaaaggggatttgattctgttaggattcccataagatggtcagcacatatatccgaaaagccaccatatgatatt gacaggaatttcctcgaaagagttaaccatgttgtcgatagggctcttgagaataatttaacagtaatcatcaata cgcaccattttgaagaactctatcaagaaccggataaatacggcgatgttttggtggaaatttggagacagattgc aaaattctttaaagattacccggaaaatctgttctttgaaatctacaacgagcctgctcagaacttgacagctgaa aatggaacgcactttatccaaaagtgctcaaagttatcagggagagcaatccaacccggattgtcattatcgatgc tccaaactgggcacactatagcgcagtgagaagtctaaaattagtcaacgacaaacgcatcattgtttccttccat tactacgaacctttcaaattcacacatcagggtgccgaatgggttaatcccatcccacctgttagggttaagtgga atggcgaggaatgggaaattaaccaaatcagaagtcatttcaaatacgtgagtgactgggcaaagcaaaataacgt accaatctttcttggtgaattcggtgcttattcaaaagcagacatggactcaagggttaagtggaccgaaagtgtg agaaaaatggcggaagaatttggattttcatacgcgtattgggaattttgtgcaggatttggcatatacgatagat ggtctcaaaactggatcgaaccattggcaacagctgtggttggcacaggcaaagagtaa 15 MEQSVAESDSNSAFEYNKMVGKGVNIGNALEAPFEGAWGVRIEDEYFEIIKKRGFDSVRIPIRWSAHISEKPPYDI DRNFLERVNHVVDRALENNLTVIINTHHFEELYQEPDKYGDVLVEIWRQIAKFFKDYPENLFFEIYNEPAQNLTAE KWNALYPKVLKVIRESNPTRIVIIDAPNWAHYSAVRSLKLVNDKRIIVSFHYYEPFKFTHQGAEWVNPIPPVRVKW NGEEWEINQIRSHFKYVSDWAKQNNVPIFLGEFGAYSKADMDSRVKWTESVRKMAEEFGFSYAYWEFCAGFGIYDR WSQNWIEPLATAVVGTGKE 16 ggatccaccatgagggtgttgctcgttgccctcgctctcctggctctcgctgcgagcgccaccagcggcgtggacc cgttcgagaggaacaagatcctgggcaggggcatcaacatcggcaacgccctggaggccccgaacgagggcgactg gggcgtggtgatcaaggacgagtacttcgacatcatcaaggaggccggcttcagccacgtgagaatcccgatcagg tggagcacccacgcccaggccttcccgccgtacaagatcgaggacaggttcttcaagagggtggacgaggtgatca acggcgccctgaagaggggcctggccgtggtgatcaaccagcaccactacgaggagctgatgaacgacccggagga gcacaaggagaggttcctggccctgtggaagcagatcgccgacaggtacaaggactacccggagaccctgttcttc gagatcctgaacgagccgcacggcaacctgaccccggagaagtggaacgagctgctggaggaggccctgaaggtga tcaggagcatcgacaagaagcacaccatcatcatcggcaccgccgagtggggcggcatcagcgccctggagaagct gagggtgccgaagtgggagaagaacgccatcgtgaccatccactactacaacccgttcgagttcacccaccagggc gccgagtgggtggagggcagcgagaagtggctgggcaggaagtggggcagcccggacgaccagaagcacctgatcg aggagttcaacttcatcgaggagtggagcaagaagaacaagaggccgatctacatcggcgagttcggcgcctacag gaaggccgacctggagagcaggatcaagtggaccagcttcgtggtgagggaggccgagaagaggaggtggagctgg gcctactgggagttctgcagcggcttcggcgtgtacgacaccctgaggaagacctggaacaaggacctgctggagg ccctgatcggcggcgacagcatcgagagcgagaaggacgagctgtgagagctca 17 MRVLLVALALLALAASATSGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEYFDIIKEAGFSHVRIPIRWST HAQAFPPYKIEDRFFKRVDEVINGALKRGLAVVINQHHYEELMNDPEEHKERFLALWKQIADRYKDYPETLFFEIL NEPHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISALEKLRVPKWEKNAIVTIHYYNPFEFTHQGAEW VEGSEKWLGRKWGSPDDQKHLIEEFNFIEEWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRRWSWAYW EFCSGFGVYDTLRKTWNKDLLEALIGGDSIESEKDEL 18 ggatccaccatgagggtgttgctcgttgccctcgctctcctggctctcgctgcgagcgccaccagcggcgtggacc cgttcgagaggaacaagatcctgggcaggggcatcaacatcggcaacgccctggaggccccgaacgagggcgactg gggcgtggtgatcaaggacgagttcttcgacatcatcaaggaggccggcttcagccacgtgagaatcccgatcagg tggagcacccacgcccaggccttcccgccgtacaagatcgagccgagcttcttcaagagggtggacgaggtgatca acggcgccctgaagaggggcctggccgtggtgatcaacatccaccactacgaggagctgatgaacgacccggagga gcacaaggagaggttcctggccctgtggaagcagatcgccgacaggtacaaggactacccggagaccctgttcttc gagatcctgaacgagccgcacggcaacctgaccccggagaagtggaacgagctgctggaggaggccctgaaggtga tcaggagcatcgacaagaagcacaccgtgatcatcggcaccgccgagtggggcggcatcagcgccctggagaagct gagggtgccgaagtgggagaagaacgccatcgtgaccatccactactacaacccgttcgagttcacccaccagggc gccgagtgggtgccgggcagcgagaagtggctgggcaggaagtggggcagcccggacgaccagaagcacctgatcg aggagttcaacttcatcgaggagtggagcaagaagaacaagaggccgatctacatcggcgagttcggcgcctacag gaaggccgacctggagagcaggatcaagtggaccagcttcgtggtgagggaggccgagaagaggggctggagctgg gcctactgggagttctgcagcggcttcggcgtgtacgacccgctgaggaagcagtggaacaaggacctgctggagg ccctgatcggcggcgacagcatcgagagcgagaaggacgagctgtgagagctca 19 MRVLLVALALLALAASATSGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWST HAQAFPPYKIEPSFFKRVDEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIADRYKDYPETLFFEIL NEPHGNLTPEKWNELLEEALKVIRSIDKKHTVIIGTAEWGGISALEKLRVPKWEKNAIVTIHYYNPFEFTHQGAEW VPGSEKWLGRKWGSPDDQKHLIEEFNFIEEWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRGWSWAYW EFCSGFGVYDPLRKQWNKDLLEALIGGDSIESEKDEL 20 ggatccaccatgagggtgttgctcgttgccctcgctctcctggctctcgctgcgagcgccaccagcggcgtggacc cgttcgagaggaacaagatcctgggcaggggcatcaacatcggcaacgccctggaggccccgaacgagggcgactg gggcgtggtgatcaaggacgagtacttcgacatcatcaaggaggccggcttcagccacgtgagaatcccgatcagg tggagcacccacgcccaggccttcccgccgtacaagatcgaggacagcttcttcaagagggtggacgaggtgatca acggcgccctgaagaggggcctggccgtggtgatcaacatccaccactacgaggagctgatgaacgacccggagga gcacaaggagaggttcctggccctgtggaagcagatcgccgacaggtacaaggactacccggagaccctgttcttc gagatcctgaacgagccgcacggcaacctgaccccggagaagtggaacgagctgctggaggaggccctgaagtgat caggagcatcgacaagaagcacaccgtgatcatcggcaccgccgagtggggcggcatcagcgccctggagaagctg agggtgccgaagtgggagaagaacgccatcgtgaccatccactactacaacccgttcgagttcacccaccagggcg ccgagtgggtgccgggcagcgagaagtggctgggcaggaagtggggcagcccggacgaccagaagcacgtgatcga ggagttcaacttcatcgaggagtggagcaagaagaacaagaggccgatctacatcggcgagttcggcgcctacagg aaggccgacctggagagcaggatcaagtggaccagcttcgtggtgagggaggccgagaagaggggctggagctggg cctactgggagttctgcagcggcttcggcgtgtacgacccgctgaggaagcagtggaacaaggacctgctggaggc cctgatcggcggcgacagcatcgagagcgagaaggacgagctgtgagagctca 21 MRVLLVALALLALAASATSGVFPFERNKILFRFINIGNALEAPNEGDWGVVIKDEYFDIIKEAGFSHVRIPIRWST HAQAFPPYKIEDSFFKRVDEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIADRYKDYPETLFFEIL NEPHGNLTPEKWNELLEEALKVIRSIDKKHTVIIGTAEWGGISALEKLRVPKWEKNAIVTIHYYNPFEFTHQGAEW VPGSEKWLGRKWGSPDDQKHVIEEFNFIEEWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRGWSWAYW EFCSGFGVYDPLRKQWNKDLLEALIGGDSIESEKDEL 22 MAQTCLTSPQTGFHNGFFYSFWKDSPGTVNFCLLEGGRYTSNWSGINNWVGGKGWQTGSRRNITYSGSFNTPGNGY LALYGWTTNPLVEYYVVDSWGSWRPPGSDGTFLGTVNSDGGTYDIRRAQRVNAPSIIGNATFYQYWSVRQSKRVGG TITTGNHFDAWASVGLNLGTHNYQIMATEGYQSSGSSDITVS 23 atggcccagacctgcctcacgtcgccccaaaccggctttcacaatggcttcttctattccttctggaaggacagtc cgggcacggtgaattttttgcctgttggagggcggccgttacacatcgaactggagcggcatcaacaactgggtgg gcggcaagggatggcagaccggttcacgccggaacatcacgtactcgggcagcttcaatacaccgggcaacggcta cctggcgctttacggatggaccaccaatccactcgtcgagtactacgtcgtcgatagctgggggagctggcgtccg ccgggttcggacggaacgttcctggggacggtcaacagcgatggcggaacgtatgacatctatcgcgcgcagcggg tcaacgcgccgtccatcatcggcaacgccacgttctatcaatactggagcgttcggcagtcgaagcgggtaggtgg gacgatcaccaccggaaaccacttcgacgcgtgggccagcgtgggcctgaacctgggcactcacaactaccagatc atggcgaccgagggctaccaaagcagcggcagctccgacatcacggtgagttga

Fiber Length Reduction Enhancement Agent

As shown herein, it is found that the copolymers of acrylamide and acryloxyethyl-trimethylammonium chloride are exceptionally effective in working to effect enzymatic fiber length reductions. These copolymers include, for example, commercially-available BASF polymers, such as Percol® (e.g., Percol® 3232L, Percol® 3035, and others), Organopol® (e.g., Organopol®5685, Organopol®6465, Organopol®6485, and others), Zetag polymers, employed at dosages less than about 10 kilogram (of agent) per ton of pulp (kg/Ton), less than about 5 kg/Ton, about 0.2 kg/Ton to about 5 kg/Ton, or about 1 kg/Ton to about 5 kg/Ton. At doses higher than 10 kg/Ton pulp, these copolymers usually have a negative effect on the enzymatic refining in the present disclosure. Copolymers of acrylamide and diallyldimethylammonium chloride can also be used, at doses from about 0.2 kg/Ton to about 5 kg/Ton.

Another agent useful as a length reduction enhancer are the polymer or copolymers of vinylamines (or comprising vinylamines). These include, for example, BASF's commercially available polymers, such as Xelorex™ (e.g., Xelorex™ RSI300, which is a highly hydrolyzed poly n-vinylformamide with >90% amine groups). As shown herein, these agents in combination with enzymatic treatment produce surprisingly good effect on fiber length reduction, on refined smoothness, or on refined bulk at given freeness. The dosages required are usually higher than 5 kg/Ton or 10 kg/Ton to demonstrate the effect.

Another agent useful as a length reduction enhancer is free glyoxal. As shown herein, free glyoxal in combination with enzymatic refining produces surprisingly improved beneficial effect fiber length reduction. Suitable dosages of free glyoxal include from 0.2 kg/Ton and 1 kg/T, or from 1 kg/Ton and 5 kg/Ton. Optionally, the free glyoxal can be mixed with the copolymers of acrylamide and acryloxyethyl-trimethylammonium chloride or the copolymers of acrylamide and diallyldimethylammonium chloride discussed above, or can be mixed with the polymers or co-polymers comprising vinylamines. The free glyoxal can optionally be added in combination with glyoxal peroxidase enzymes.

In general, partially or completely hydrolyzed polymeric n-vinylformamide, or polymeric n-vinylacetamide, polymeric n-vinylimides such as polymeric n-vinylsuccinimide, preferably with greater than 75% degree of hydrolysis, or greater than 85% degree of hydrolysis, can be used in the disclosed methods. Also included are copolymers of n-vinylformamide and vinylamine or its hydrochloric salts, preferably with >75% vinylamine. Other copolymers include co-polymers of n-vinylformamide and diallyldimethylammonium chloride, copolymers of n-vinylformamide and acrylamide, all with various degrees of free amine groups. Various polyallylamimes are also included. Polyvinylamines generated by any methods, including by Hoffman degradation methods from polyacrylamides, are useful for the methods disclosed herein.

Optional Chemical Agents

Other chemical agents, some of which may also have some positive effect on enzymatic fiber length reduction but with less dose-effectiveness than the fiber length reduction enhancement agents discussed above, can be listed here. They can in principle be used as well, but may also be used together as further additives to the present disclosure.

Exemplary other chemical agents include g-Pam (glyoxalated polyacrylamide with low levels of free glyoxals) which can be used to increase tensile strength or wet strength, though having little effect on enzymatic fiber length reductions. Also included is polyDADMAC, which is a cationic retention polymer with very limited effect. Also included is polyethylenimines, though having some positive effect but with less dose-effectiveness and with yellowing side effects.

Other papermaking additives that can be further applied, include other copolymers of diallyldimethyl ammonium chloride (DADMAC), copolymers of vinyl pyrrolidone (VP) with quaternized diethylaminoethylmethacrylate (DEAMEMA), cationic polyurethane latex, cationic polyvinyl alcohol, polyalkylamines, dicyandiamid copolymers, amine glycigyl addition polymers, poly[oxyethylene (dimethyliminio) ethylene (dimethyliminio) ethylene] dichlorides, poly (hexamethylene biguanide hydrochloride) (i.e. PHMB), cationic metal ions, such as water-soluble aluminum salts, calcium salts, and zirconium salts; and these bound ions can act as active complexing sites for sizing and other papermaking chemicals; and cationic dendrimers, such as PAMAM (polyamidoamine) dendrimers with amino surface groups, and polypropylenimine dendrimers with amino surface groups. They also include cross-linking agents, such as cationic starches, dialdehyde starches, dialdehyde maltodextrins, multi-functional carboxylic acids, such as 1,2,3,4-butanetetracarboxylic acid (BTCA), Poly (maleic acid), (PMA), poly(itaconic acid), citric acid (preferably with catalysts), and water-dispersible or water-soluble bi-, multifunctional carbodiimide and/or polycarbodiimide such as 1,6-hexamethylene bis(ethylcarbodiimide); 1,8-octamethylene bis(ethylcarbodiimide); 1, 10-decamethylene bis(ethylcarbodiimide); 1,12 dodecamethylene bis(ethylcarbodiimide); PEG-bis(propyl(ethylcarbodiimide)); 2,2′-dithioethyl bis(ethylcarbodiimde); 1,1′-dithio-p-phenylene bis(ethylcarbodiimide); and 1,1′-dithio-m-phenylene bis(ethylcarbodiimide), during papermaking or fibrous network forming.

Further treatment of the modified pulp fiber of the disclosed methods may be internal sizing on fibers such as by AKD sizing agents, or ASA sizing agents. Further treatment may also be surface treatment on the formed paper webs, such as by starch surface sizing, and by non-reactive sizing agents, for instance, without limitation, BASOPLAST® 335D non-reactive polymeric surface size emulsion from BASF, FLEXBOND® 325 emulsion of a co-polymer of vinyl acetate and butyl acrylate from Air Products and Chemicals (Allentown, Pa.), and PENTAPRINT® non-reactive sizing agents as from Solenis (formerly Ashland Water Technologies, Wilmington, Del.).

Wood and Non-Wood Pulp Fibers

As used herein, “pulp fiber” refers to both wood pulps and nonwood pulps. Wood pulp fibers include those derived from hardwood trees, softwood trees, or a combination of hardwood and softwood trees prepared for use in a papermaking furnish by any known suitable digestion/pulping, refining, or bleaching operations. For example, the pulps may be any types, such as but not limited to, mechanical pulps, thermo-mechanical pulps, CTMP, RMP, APMP, ground wood pulps, soda pulps, soda-AQ pulps, steam explosion or aqueous water extraction pulps, green liquor pulps, sodium carbonate pulps, ammonia pulping, various variants of Kraft pulps, pre-hydrolysis Kraft pulps, various sulfite pulps, acid sulfite pulps, neutral sulfite pulps, NSSC pulps, polysulfide pulps, various solvent pulping pulps, alcohol pulps, alcohol-SO₂ pulps, ASAM pulps, and pulps from any other known pulping or defibrization methods.

The term “hardwood pulps” as used herein refers to fibrous pulp derived from the woody substance of deciduous trees (angiosperms), whereas “softwood pulps” are fibrous pulps derived from the woody substance of coniferous trees (gymnosperms).

Useful pulp fibers may also be provided from non-woody herbaceous plants including, but not limited to, kenaf, hemp, jute, flax, sisal, abaca, wheat straw, rice straw, bagasse, bamboo, corn stocks, corn cobwebs, corn kernel fibers, peanut shells, ramie, seaweed, algae, sugar beet, orange peel, orange (fruit) fibers, lemon peel, switchgrass, and cotton linters. Any bleached pulps, partially or semi-bleached pulps, unbleached pulps, high Kappa pulps, dissolving pulps, fluff pulps, sawdust pulps, are included. Bleached or unbleached recycled pulps, as well as deinked pulps may also be utilized in the process of this disclosure. The pulp may have been subjected to any treatment history that is normal in pulping and bleaching or may be intentionally modified as for example by water/steam treatment, ammonia pulping (with or without explosion), or caustic extraction of chips before pulping, acid hydrolysis of chips or pulps, enzyme (cellulases or hemicellulases) hydrolysis of pulps or chips, oxygen delignification, and “cold-soda” treatment of chips or pulps. Any bleaching chemistry as is known in the pulp and paper industry or academia, is included.

Intra-Crystalline Swelling Agents for Cellulose

The present disclosure can also be used in combination with any intra-crystalline swelling agents that can effectively swell the cellulosic fibers. This is particularly useful when used in combination with hyperthermostable cellulases, hemicellulases, and laccases. Two major classes of intra-crystalline cellulose swelling agents are: ionic liquids, and select organic solvents. Some of the intra-crystalline cellulose swelling agents can at certain conditions act as dissolution solvents for cellulose. Accordingly, such intra-crystalline swelling agents are only used at dosages (in aqueous phase) or reaction conditions sufficient to incur intra-crystalline swelling of cellulosic fibers, but not sufficient for cellulose dissolutions.

Examples of such intra-crystalline swelling agents include ionic liquids (ILs). Ionic liquids consist of a large organic cation part and the anion part. The cations of ILs may usually be based on imidazolium, pyridinium, pyrazolium, pyrrolidinium, triazolium, thiazolium, phosphonium, ammonium, quanidinium, and cholinium ions. In the present disclosure, ILs based on imidazolium, ammonium, quanidinium, and pyridinium cations are included, with all applicable anions. Specific ILs examples included in the present disclosure include (but not limited to): I-ethyl-3-methyl imidazolium acetate, 1-butyl-3-methyl imidazolium chloride, 1-allyl-3-methyl imidazolium chloride, 1-N-butyl-2,3-dimethyl imidazolium chloride, 1-N-2,3-dimethyl imidazolium bromide, 1-butyl-3-methyl imidazolium acetate, 1-allyl-3-methyl imidazolium formate, N-ethyl-N-methyl imidazolium methylphosphonate, 3-methyl-N-butyl pyridinium chloride, benzyldimethyl (tetradecyl) ammonium chloride, and 1,1,3,3-tetramethyl quanidinium acetate. One of the preferred ILs is 1-ethyl-3-methyl imidazolium acetate. Another preferred is 1-butyl-3-methyl imidazolium acetate. Another intracrystallite swelling agent, N-methylmorpholine-N-oxide (NMMO) is also listed here. The amount of ILs used is based on the conditions to cause intracrystalline swelling, yet not sufficient to dissolve cellulose. For example, in one option, 0.2% to 50% ILs can be applied. The treatment temperature depends on the hyperthermostable enzymes, usually between 70° C. to 120° C., between 75° C. to 120° C., or between 80° C. to 105° C.

Examples of such intra-crystalline cellulose swelling organic solvents (in the aqueous phase) include, but not limited to: formamide, DMSO, N-alkylated lactams, N-alkylated ureas, N-alkylated amides, and particularly tetramethyl urea, tetraethyl urea, dimethylpropyleneurea, dimethylethyleneurea, N-cyclohexylpyrrolidinone, N-methyl pyrrolidinone, N-ethyl pyrrolidone, N-octyl pyrrolidone, dimethyl acetamide and hexamethyl phosphoric amide. They also include various polyol or glycol ethers, such as cellusolve (2-Ethoxyethanol), butyl cellusolve (ethylene glycol monobutyl ether), butyl carbitol (diethylene glycol monobutyl ether), ethyl carbitol, propylene glycol monomethyl ether, dipropylene glycol monomethyl ether.

Paper, Pulp, and Products

The methods of the present disclosure provide a modified pulp useful for making any paper and paper web materials. Thus, the disclosure includes products comprising the modified pulp. Paper and paper web products comprising modified pulp of the disclosure include various paper grades, uncoated or coated, unbleached, semi-bleached, or fully bleached paper grades, printing and writing papers of any sorts, uncoated free sheet, surface sized papers, high yield papers (containing mechanical or chemi-mechanical pulps), newsprint, LWC, film coated mechanical grades, surface treated mechanical grades, paper boards of any types, uncoated board, coated board, white board, brown boards, SUS, SBS, multi-layered boards, FBB, folding cartons, linerboards, kraft papers, sack papers, wrapping papers, release papers, silicone papers, recycled brown papers, recycled white papers, deinked papers, paper filters, mercerized papers, base papers for polymer impregnation, base papers for composites, TAPPI paper plates, paper cups, impregnation papers such as Formica, sulfite papers, medium papers, and corrugated boards, tissues, towels, napkins, wipes, nonwovens, personal care pads, cigarette papers, fluff pulp rolls, dissolving pulp rolls and sheets, any cellulose-containing paper webs used as printing or digital printing media.

The methods of the present disclosure can also be used to produce pulp or paper materials that can be further converted into specialty materials, such as cellulose foams, cellulose films, microcrystalline cellulose, microfibrillated cellulose, nanocellulose or nanofibrillated cellulose materials from either lignin-free or lignin-containing (wood or non-wood) pulps processed by the present methods. The disclosure therefore includes products comprising the modified pulp, wherein the products include cellulose foams, cellulose films, microcrystalline cellulose, microfibrillated cellulose, nanocellulose or nanofibrillated cellulose materials from either lignin-free or lignin-containing (wood or non-wood) pulps processed by the disclosed methods.

EXAMPLES

The products, compositions and methods of making and using are further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the products, compositions and methods of the disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

The following materials were employed in the examples.

Cellulase A is a commercially-available, powdered enzyme product obtained from Chrysosporium lucknowense/Myceliophthora thermophilia and which has predominantly endoglucanase activities.

Cellulase B is a hyperthermostable cellulase having the amino acid sequence shown in SEQ ID NO: 6. It is commercially available as a dilute enzyme solution, with endoglucanase activities, usually assayed at 80° C. Given the dilute concentration of the enzyme solution, high doses were used in the experimental examples.

Xelorex™ RS 1300 (BASF Corporation, Florham Park, N.J.) is a highly hydrolyzed polyvinyl formamide with >90% amine groups.

Percol® 3232L (BASF Corporation, Florham Park, N.J.) is copolymer of acrylamide and acryloxyethyl-trimethylammonium chloride.

The following experimental procedures were employed in the examples.

PFI refining or beating, is a standard lab scale pulp refiner used in pulp and paper industry, as described by TAPPI Standard T248.

TAPPI Handsheets were made according to TAPPI Standard T205 and T220.

TAPPI Freeness was tested according to TAPPI Standard T227.

TAPPI Tensile Strength and index was tested according to TAPPI Standard T494.

TAPPI Sheffield Smoothness was tested according to TAPPI Standard T538.

TAPPI Tear Strength was tested according to TAPPI T414. In this study, T414 was also used for DSF (Dynamic Sheet Former) Sheet CD tear test. (“CD”=“cross-machine direction.”

TAPPI Bursting Strength was tested according to TAPPI T403 om-10.

Valley Beater refining was done under a total load of weighing plates of 12 kg, and for each refining experiment, a total of 400 o.d. gram of pulp was refined at 2% consistency, at various refining/beating times. (o.d.=oven-dry).

Dynamic Sheet Former (DSF) sheet preparation was made at 70 g/m² handsheets, with thickstock of 2% consistency diluted to thinstock of 0.5% consistency with tap water. The DSF settings were: Drum Speed=1200 m/min, Pump Speed=500 rds/min, Spray Nozzle: 2515. The formed DSF hand sheets were pressed at 60 psi and dried on a drum dryer at 240° F. for five minutes. The dried sheets were then conditioned overnight at standard laboratory conditions of: 72° F.±5° F., 50%±5% R.H. The conditioned handsheets were then tested.

Fiber Length Distributions were tested on an FQA Fiber Length Analyzer, by OpTest, Model LDAO2.

Paper bulk was measured according to TAPPI Standard T411.

Example 1 PFI Enzyme Refining

A bleached US southern pine Kraft pulp was treated with Cellulase A at a dosage of 0.2 kg enzyme/ton pulp (kg/T), and at a dosage of 2.0 kg/T, at papermaking temperature of 55° C., 4% pulp consistency and pH 6, for 1 hour. The enzyme-treated pulp was then subjected to standard TAPPI PFI refining (gap 0.2 mm), from which TAPPI standard 1.2 gram handsheets were made and tested. The results are shown in Table 2. The freeness and tensile index data, as a function of energy (PFI revolutions), are plotted in FIGS. 1 and 2, respectively. In Table 2, “NA” means “not tested.”

TABLE 2 PFI Refining and TAPPI Sheet FQA, L- weighted Sheffield Avg Fiber PFI Tensile Smooth- Length (Revolu- Freeness Index ness (LWAFL; tions) (CSF) (N · m/g) (ml/min) mm) Control 0 742 30.8 2831 2.58 3000 529 76.8 2196 2.57 5400 398 88.7 1810 2.55 7400 300 85.4 1474 NA 11200 197 90.6 1023 2.4 0.2 kg/T 0 661 41.4 2674 2.57 Cellulase 2200 535 73.4 2298 NA A Treated 5000 385 84.2 1873 NA 7600 285 83.0 1355 NA 11000 212 86.9 1098 NA   2 kg/T 0 686 40.5 2741 2.64 Cellulase 1700 526 71.5 2428 2.44 A Treated 2800 436 76.5 2094 2.36 4800 299 81.2 1500 2.31 8000 195 83.4 1196 2.24   2 kg/T 0 769 34.9 2825 2.62 Cellulase 4000 581 75.5 2074 2.29 A Treated, 6600 414 79.4 1168 2.23 Co-refined 9000 280 83.7 876 2.15 with 10 11400 200 86 847 NA kg/T Xelorex ™

FIG. 1 illustrates that enzyme treatment on southern pine pulp can reduce refining energy. FIG. 2 shows that for unrefined pulp, or for low levels of refining, enzyme treatment may enhance paper strength development. However, at higher refining levels (or lower freeness levels), the intrinsic strength is not improved vs. control softwood.

In practice, these results can be exploited to reduce refining energy, de-bottle-neck refiner capacity, and/or improve strength for papers of certain freeness targets. It should be noted that for recycled furnish or once-dried pulps, the beneficial effect would typically be more pronounced due to the enzyme's actions on removing the “hornification” caused by drying, finishing and utilizations. Pulp species and the types/intensity of various refiners would also have dramatic effects as well.

Example 2 Enzyme Treatment with Co-Refining with Xelorex™

In this example, enzyme treatment on southern pine Kraft pulp was conducted in the same conditions as in Example 1 with 2 kg/T cellulose A. After enzyme treatment, Xelorex™ RS 1300 was added to the pulp at dose of 10 kg/T, and the pulp was co-refined. Sheffield smoothness and fiber length distribution (tested on FQA Fiber Length Analyzer) were tested. The PFI refining and testing results are also listed in Table 2. Smoothness as a function of energy (PFI revolutions) and as a function of freeness (CSF) are plotted in FIGS. 3 and 4, respectively. Fiber length distribution as a function of freeness is plotted in FIG. 5.

FIG. 3 and FIG. 4 indicate that cellulase enzyme treatment followed by co-refining with Xelorex™ unexpectedly improved smoothness (reduced Sheffield values) after co-refining. This substantially better smoothness after co-refining was also shown clearly at the given freeness vs control refining. In comparison, cellulase enzyme treatment alone (without Xelorex™) at either dosage tested did not show improvement in smoothness when compared at the same freeness (FIG. 4). However, the improvement of smoothness for the cellulase enzyme treatment followed by co-refining with Xelorex™ could be accelerated or facilitated by the cellulase enzyme refining.

The data in FIGS. 3 and 4 suggest that the controlled fiber interior modification (“damage”) as exemplified by fiber length reduction due to enzymes, demonstrates a synergistic effect with Xelorex™ co-refining. The synergy is also evident in the fiber length reductions data depicted in FIG. 5, supporting the apparent synergism.

It is expected that this refining synergy in smoothness can be exploited to improve paper surface and printing properties of coarse fibers such as southern pine fibers, southern hardwood fibers, or other coarse fibers (such as Douglas fir and hemlock). It may also be used to alleviate paper surface linting, and hardwood vessel picking issues.

Example 3 Bulk Improvement with Energy Savings on Refining

Enzyme treatment was carried out on a re-slurred commercial bleached southern pine Kraft pulp at 48 C, 4% consistency, pH 6.6, for 1 hour, with an enzyme dosing of 2 kg/T Cellulase A. After enzyme treatment, the pulp was diluted to 2% consistency and refined by Valley-Beater at various beating time (minutes) to various freeness levels. The enzymatic refined pulp was then blended with 10 kg/T Xelorex™ RS1300, and made into DSF (Dynamic Sheet Former) sheets of 70 g/m² target, dried, conditioned, and tested for paper properties. The control pulp was not enzyme treated and was not blended with Xelorex™, prior to being made into DSF sheets. The results normalized at 70 g/m² are listed in Table 3.

TABLE 3 Valley Beater and DSF Sheets Valley- Beater MD Mullen CD Time Freeness Bulk Tensile Burst Tear (minutes) (CSF) (cc/g) (kgf/mm) (psi) (gf) Control 0 742 2.134 0.359 25.5 125.07 10 590 1.503 0.871 55.8 95.91 20 290 1.310 0.943 63.0 79.54  2 kg/T 0 740 2.082 0.567 45.6 138.42 Cellulase A 10 475 1.703 0.910 48.8 82.16 treated and 20 130 1.452 1.051 50.3 61.53 refined, then blended with 10 kg/T Xelorex ™

FIG. 6, FIG. 7 and FIG. 8 illustrate that while refining energy saving was achieved by enzyme, the paper bulk was significantly increased as well after refining. It is surprising that paper bulk increase was also shown when compared at given refined freeness. However, it should be noted that, the paper bulk before refining did not show increased paper bulk. It is believed the synergy of the enzymatic refining process with the Xelorex™ generated the bulk development.

Contrary prior art assertions that cellulase enzymes combined with polyvinylamines would improve paper strength substantially, FIG. 9 shows that there was very little change on tensile strength at same refined freeness in the common papermaking freeness range of 400-600 csf, except for the unrefined pulp. Unrefined pulp refers to pulp with no refining, i.e., at 0 minutes. This is also shown by tear strength (see FIG. 10) and burst strength (see Table 3), which were actually reduced after refining (except for the unrefined pulp). These findings are consistent with the intentional and controlled fiber interior damage as exemplified by fiber length reductions during the enzyme refining.

Example 4 Hyperthermostable Cellulase Enzyme—No Effect on Refining Energy at Papermaking Temperatures (Comparative)

A commercially-available dilute enzyme formulation comprising a hyperthermostable cellulase, Cellulase B, was used for this example. The enzyme treatment was conducted on a virgin bleached southern pine pulp under the same conditions as Example 1, at a typical papermaking (paper machine) temperature of 55° C., pH 6, and at 4% consistency, for 1 hour. The treated pulp was then refined at 2% consistency by a Valley Beater. The refining/beater data are shown in Table 4 and plotted in FIG. 11. Unlike the result with Cellulase A, the hyperthermostable Cellulase B enzyme at this temperature (55° C.) showed no effect on refining energy, despite the very high enzyme dosing (20 kg/ton) of Cellulase B used.

TABLE 4 Valley Beating Time Control Refining 55° C. Cellulase B Refining 55° C. (minutes) Freeness (CSF) Freeness (CSF) 0 740 730 10 590 610 20 290 310 30 170 130

Example 5 Enabling the Use of Hyperthermostable Cellulase Enzymes in Papermaking

As shown in Example 4, the hyperthermostable enzyme Cellulase B did not effectively function at typical papermaking temperatures. Therefore, the function of Cellulase B was tested at higher temperatures in this example, as well as in Example 8. In this experiment, the enzyme treatment was conducted at 70° C., pH6, for 4 hours, on another source of southern pine pulp. Conditions comparable to these experimental conditions can actually be obtained/achieved, for instance, upstream at pulp mill stages, bleach plant stages, brown HD towers, or bleached HD towers of a commercial paper mill. The control pulp (no enzyme treatment) and the various enzyme-treated pulps were refined by a Valley Beater as before. Freeness and Fiber Length Distribution (FQA fiber average length) were assessed. The data are shown in Table 5, and graphed in FIGS. 12 and 13. The resulting refining/beater curves, listed side-by side with Cellulase A (55° C., 4 hours), illustrate that Cellulase B actually works very well at these conditions (70° C., pH6). FIG. 12 reveals that hyperthermostable Cellulase B can be effective in refining. FIG. 13 illustrates the effectiveness of hyperthermostable Cellulase B in fiber length reduction.

Table 5 summarizes the Valley-Beater refining data of these experiments, including also the effect of previously demonstrated fiber length reduction enhancing agents, such as Percol® 3232L and Xelorex™ RSI300.

TABLE 5 FQA Fiber Average Final Length Valley Beater Freeness, (L-weighted), Time, minutes CSF mm Control Refining (No 0 635 2.37 Enzyme) 5 610 10 570 15 515 2.21 20 430 25 360 2.01 30 280 35 210 40 165 Cellulase B at 70° C., 10 0 610 kg/T 5 590 10 550 15 460 2.10 20 375 2.02 25 295 1.89 30 210 1.79 35 100 Cellulase B at 70° C., 15 0 625 kg/T 5 590 10 460 1.89 15 350 1.59 20 210 1.37 25 155 Cellulase B at 70° C., 20 0 550 2.37 kg/T 5 220 0.79 Cellulase A at 55° C., 0.2 0 650 kg/T 5 630 10 530 15 430 2.06 20 320 1.86 25 240 30 170 Cellulase A at 55° C., 0.4 0 625 kg/T 5 550 10 370 1.75 15 215 1.64 20 120 Cellulase A at 55° C., 0 575 2.44 2 kg/T 5 220 0.62

One of the unique benefits of using thermostable enzymes that work at pulp mill or bleach plant temperatures, which have higher temperatures than the papermaking/paper mill temperatures, is that the enzyme-treated pulp (with or without washing), when transferred to the papermaking stages (stock preparation or paper machine), will display little or no enzyme active function at paper machine white water system temperatures, therefore having no long term side effects on the papermaking systems. Unlike the case of normal (mesophilic) cellulase (such as Cellulase A), the residual hyperthermostable enzyme, if still present in the pulp, would be inhibited during papermaking temperatures, and would be inactivated/denatured by the drying process. Another option is to conduct cellulase enzyme treatment (both normal cellulase embodiment, and thermostable cellulase embodiment) at Bleached HD Towers (at the end of the bleach plant operation) which would also optionally provide extended retention (and reaction) time, thus enabling reduced enzyme doses.

Example 6 Converting Southern Pine Fiber Morphology by Cellulase Enzymes (Valley Beater Experiment)

This example illustrates the reduction of southern pine fiber length (length-weighted average length) from 2.3 mm-2.5 mm down to 0.6 mm-1.8 mm, by a low intensity lab refining device such as the Valley Beater, while maintaining a pulp freeness between 400 CSF to 650 CSF range (preferred papermaking region), and, preferably for paper board, in the 500 CSF to 650 CSF range. It should be noted that these are relative comparisons, as disc refiners would have different values although the trend is expected to be comparable. (The strategy with disc-refiners or Conflo refiners is discussed in Example 9).

The resulting fiber length numbers in this Example are summarized with the enzyme and refining data in Table 5. The fiber length data are plotted in the FIG. 14. These data show, relatively speaking, that while it is possible for enzyme refining alone to reach regions of the target range, high dosing of enzymes are required. High enzyme dosing may not be preferred as it may impact not just economics, but also possible excessive degradations of paper strength properties at such high enzyme dosages.

Example 7 Converting Southern Pine Fiber Morphology with Optimal Enzyme Dose in Synergy with Fiber Length Reduction Enhancing Agents

This example demonstrates the use of optimal (economical) dose of enzyme, with energy savings, fiber length reduction to target freeness range, in combination (by co-refining) with a fiber length reduction agent (Xelorex™ or Percol®). In addition, this example demonstrates the use of enzyme, with the fiber length reduction agent (Xelorex™ or Percol®) added post-refining.

The data are shown in Table 6. Again, these data are relative comparisons using Valley Beater as refiners. The values based on disc-refiners would be different although the trend is expected to be comparable.

TABLE 6 Valley FQA Fiber Beater Final Average Time, Freeness, Length (L- minutes CSF weighted), mm Control Co-refined with 0 675 Percol ® 2 kg/T (No enzyme) 5 670 10 630 15 555 2.20 20 460 25 380 2.07 30 305 35 260 40 190 Cellulase A 0.4 kg/T Treated, 0 735 and Co-refined with 2 kg/T Percol ® 5 645 10 520 1.91 15 340 1.62 20 210 1.42 25 130 Cellulase A 0.4 kg/T Treated, 0 685 and Co-refined with 10 kg/T Xelorex ™ 5 605 10 470 1.70 15 325 1.42 20 195 1.19 Cellulase A 0.4 kg/T Refined 15 520 1.64 and post-added with 2 kg/T Percol ® Cellulase A 0.4 kg/T Refined 15 380 1.64 and post-added with 10 kg/T Xelorex ™

FIG. 15 and FIG. 16 show that the co-refining control (without enzyme treatment) with Percol 3232 increased refining energy consumption slightly vs. the control refining alone, while the fiber length and freeness relationship was not affected at all. However, surprisingly, the enzyme treated fiber at 0.4 kg/ton Cellulase A dosage, when co-refined with 2 kg/ton Percol® 3232 or 10 kg/ton Xelorex™ RSI300, showed synergistic effect on fiber length reduction, while still maintaining refining energy savings. Therefore, the enzyme/chemical co-refining, economically enabled low dose of enzyme treatment to reach the target fiber length reduction region, while having freeness on target as well.

FIG. 17 indicates that refining after 0.4 kg/T Cellulase A enzyme treatment, followed by addition of 2 kg/ton Percol® 3232, enabled fiber length reduction to reach the target freeness regions, while achieving energy savings delivered by Cellulase A (FIG. 15). At this enzyme dose of 0.4 kg/T, post-added Xelerox™ also showed improved fiber length reduction and freeness relationship as well when compared with 0.4 kg/ton enzyme refining alone, but it was not sufficient to reach the preferred target freeness region. Higher dosing of enzyme treatment is expected to enable reaching the preferred target freeness region. In comparison, 0.4 kg/T Cellulase A refining alone could not reach the fiber length reduction at the target freeness regions. Higher dosage (2 kg/ton) of Cellulase A treatment could enable reaching some part of the target fiber length-freeness regions, but still at lower freeness levels as compared to the combined 0.4 kg/ton Cellulase A treatment with post-added 2 kg/ton Percol® 3232.

Example 8 Hyperthermostable Cellulase B Enzyme Treatment at 85° C. on Saving Refining Energy

In this example, a commercial southern pine market Kraft pulp was re-slurried and treated with the hyperthermostable Cellulase B at dosage of 20 kg enzyme/ton pulp, at temperature of 85° C., pH 6.6, 4% consistency, for 4 hours. As a control, the southern pine slurry was also treated at 85° C., pH 6.6, 4% consistency, for 4 hours, without enzyme. Both the treated and the control pulp were then refined by a Valley Beater at time increments of 5 minutes. The results are summarized in Table 7 and plotted in FIG. 18 below.

TABLE 7 Valley Beater Time Freeness (minutes) Hyperthermostable Cellulase A Control 0 730 700 5 680 700 10 540 660 15 350 600 20 240 550 25 450 30 390 35 300

These data clearly demonstrate that hyperthermostable cellulase treatment at 85° C. resulted in substantial energy savings in pulp mechanical refining. For instance, it required 15 to about 30 minutes of Valley Beater time for the control to reach a freeness of 600 to 400 CSF. In notable contrast, it required only about 7 to about 14 minutes for the hyperthermostable enzyme-treated pulp to reach the same range of freeness.

Example 9 A Strategy of Cellulase Enzyme Refining for Converting Softwood Fiber to Hardwood Fiber Length within Papermaking Freeness Range

In this example, a strategy of cellulase enzyme refining was illustrated for converting softwood fiber into hardwood fiber length, while maintaining pulp freeness at papermaking freeness range. A northern bleached softwood Kraft pulp was re-slurried in tap water and treated with Cellulase A at dosage of 1 kg enzyme/ton of pulp at around 50° C. for 1.5 hours. The enzyme and pulp system was inactivated by 0.05% sodium hypochlorite based on the weight of the pulp (20 ppm sodium hypochlorite based on total water slurry) for 15 minutes. Refining of the treated softwood pulp was conducted on a pilot scale Sunds Defibrator Conflo JC-00 Conical Refiner (Varaoke International, Finland). The refining operation was carried out by increasing the refining intensity such as specific edge load (SEL), while maintaining the mass flow rate, rpm, and CEL (refiner plate parameter) substantially the same. The net load of energy increase was achieved by varying the plate gap size. The results are summarized in Table 8 below.

TABLE 8 Pilot Scale Refining of Softwood Treated with 1 kg/Ton Cellulase A FQA Fiber Pulp Average Length Freeness, Specific Energy (L-weighted), CSF SEL (J/m) KWh/Ton pulp mm Control 706 0 0 2.097 Refining 667 0.4 22.0 NA 635 0.8 33.6 NA 607 1.2 56.7 NA 564 1.6 72.2 2.009 517 2 86.6 NA 474 2.4 106.3 1.944 Enzyme 702 0 0 2.132 Refining, 1 598 0.13 10.1 1.654 kg/ton 526 0.25 10.2 1.311 Cellulase A 474 0.4 24.2 1.101 415 0.8 34.1 0.978 404 1.2 47.3 NA 411 1.6 67.9 NA 392 2 77.0 0.930 Enzyme 646 0.8 34.1 0.978 Refining, 1 637 2 77.0 0.930 kg/ton Cellulase A, with post- added 2 kg/ton Percol 3232

The freeness vs. specific edge load data for the control and enzyme-treated pulp are plotted in FIG. 19. FIG. 19 shows the refined freeness of enzyme treated softwood was substantially impacted by SEL, while the control (no enzyme) pulp was little affected.

The freeness vs. specific refining energy data for the control and enzyme-treated pulp are plotted in FIG. 20. FIG. 20 illustrates that the electrical energy consumption of the enzyme treated pulp was substantially reduced compared to the electrical energy consumption of the control pulp refining.

FIG. 21 is a plot of softwood fiber average length (length-weighted) vs. refined freeness data. FIG. 21 demonstrates that the enzyme-treated softwood fiber length (starting at ˜2.1 mm to 2.3 mm range) was successfully reduced by refining to the hardwood fiber length (usually 0.8 mm to 1.2 mm range), while maintaining freeness above 400 CSF. In contrast, the fiber length of control (no enzyme) refined softwood pulp was significantly less affected. In fact, the control softwood refining would decrease the freeness too much to be useful (or practically feasible) in papermaking, if significant fiber length reduction needs to be achieved. FIG. 21 also plots data that indicates the synergy of enzyme refining with post-refining-added Percol® 3232.

FIG. 22 is a plot of fiber average length (length-weighted) vs. refining energy. It further illustrates that the control refining would require too much electrical energy to reduce the fiber length significantly. In contradistinction, the enzyme-treated softwood fiber length can be converted to hardwood fiber length range with very small amount of electrical energy consumptions.

Example 10 Enzymatic Removing of Hemicellulose at 75° C. for Modified Pulp

In this example, the effect of thermostable hemicellulase enzymes, such as xylanase of SEQ ID NO: 22, in removing the hemicellulose content for generating a modified pulp is described. In general, the removal of hemicellulose content by xylanase of SEQ ID NO: 22 is more pronounced when treating on the regular pulps such as Kraft pulps of paper grades wherein the hemicellulose content is high. In this example, hemicellulose was measured by the Pentosan test (TAPPI Method T223 CM-01). With xylanase of SEQ ID NO: 22, hemicellulose removal higher than by 0.5% wt can be achieved on regular Kraft pulps. The data show that pretreatment with mechanical refining or beating, followed by xylanase treatment with xylanase of SEQ ID NO: 22, can further augment this effect, for instance, by removing more than 1% wt. of hemicellulose (Pentosan) on regular Kraft pulps. Removal of hemicellulose from dissolving pulps is much more difficult, due to the fact that dissolving pulps have very low hemicellulose content and the remaining hemicellulose inside dissolving pulp is also more resistant to the removal process by enzymes, which has historically been done with strong alkali extractions.

In this example, the data show that hyperthermostable xylanase treatment (such as by xylanase of SEQ ID NO: 22) on dissolving pulps can be further enhanced in combinations with cellulase enzymes (such as hyperthermostable Cellulose B, preferably or optionally also with intracrystalline cellulose swelling agents, such as ionic liquids). Dissolving pulp treatment by hemicellulase enzymes or by combinations of hemicellulase and cellulase enzymes can be further enhanced with pre-treatment by mechanical refining or beating as well.

Table 9 lists experimental results on the use of hyperthermostable stable enzymes for hemicellulose (or Pentosan content) removal on dissolving pulps. All the enzyme treatments (xylanase of SEQ ID NO: 22 dose 0.6 kg/ton, or Cellulase B dose 20 kg/ton), were carried out at 75° C. for 4 hours. Ionic liquid used is I-ethyl-3-methylimidazolium acetate at 10 kg/ton dose.

TABLE 9 Pentosan Pentosan (Hemi- (Hemi- cellulose) cellulose) Pulps Enzyme Treatment Conditions Content Removed Dissolving Control 3.6% Not Pulp #1 applicable Dissolving Xylanase of SEQ ID NO: 22 3.4% 0.2% wt. Pulp #1 Dissolving Xylanase of SEQ ID NO: 22 + 3.2% 0.4% wt. Pulp #1 Cellulase B Dissolving Ionic Liquid alone 3.5% 0.1% wt. Pulp #1 Dissolving Xylanase of SEQ ID NO: 22 + 3.0% 0.6% wt. Pulp #1 Cellulase B + Ionic Liquid Dissolving Control 3.2% Not Pulp #2 applicable Dissolving Xylanase of SEQ ID NO: 22 2.9% 0.3% wt. Pulp #2 Dissolving Control 2.2% Not Pulp #3 applicable Dissolving Refining pretreated, Xylanase 1.7% 0.5% wt. Pulp #3 of SEQ ID NO: 22 Dissolving Refining pretreated Xylanase 1.5% 0.7% wt. Pulp #3 of SEQ ID NO: 22, bleached

The modified pulps herein, of any sources (such as paper pulps, dissolving pulps, fluff pulps, personal care pulps, industrial use pulps, polymer impregnation pulps, etc.), can be used as pulps of upgraded performances and/or enhanced reactivity.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety for all purposes.

While the products, compositions, methods of making them, and their methods of use have been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations may be devised by others skilled in the art without departing from the true spirit and scope of the described products and methods. The appended claims are intended to be construed to include all such embodiments and equivalent variations. 

What is claimed is: 1-33. (canceled)
 34. A method of modifying papermaking pulp comprising contacting pulp comprising papermaking fibers with (a) a cellulolytic enzyme under conditions to induce fiber interior damage, and (b) a fiber length reduction enhancement agent to produce a modified pulp having: (i) a reduction of papermaking fiber length of 5% to 96% after going through a refiner or after by-passing a refiner, and (ii) a pulp freeness level of from about 200 Canadian Standard Freeness (CSF) to about 700 CSF.
 35. The method of claim 34, wherein the modified pulp is a refined pulp.
 36. The method of claim 34, wherein contacting the pulp with the fiber length reduction enhancement agent occurs preferably after contacting the pulp with the enzyme.
 37. The method of claim 34, wherein the fiber length reduction enhancement agent is selected from the group consisting of: a hydrolyzed polymer or copolymer of vinylformamide with a degree of hydrolysis greater than 70%; a copolymer of acrylamide and acryloxyethyl-trimethylammonium chloride; a copolymer of acrylamide and diallyldimethylammonium chloride; a free glyoxal; and any mixture of two of more thereof.
 38. The method of claim 34, wherein the cellulolytic enzyme is a cellulase or a cellulase-functioning hemicellulase enzyme.
 39. The method of claim 38, wherein the cellulolytic enzyme is a cellulase selected from the group consisting of: a cellulase obtained or derived from Chrysosporium lucknowense/Myceliophthora thermophilia, a cellulase obtained or derived from Humicola insolens, a cellulase obtained or derived from Aspergillus, a cellulase obtained or derived from Trichoderma, and combinations thereof.
 40. The method of claim 34, wherein the pulp comprises papermaking fiber selected from the group consisting of: a softwood fiber, a hardwood fiber, and a mixture thereof.
 41. The method of claim 34, wherein the fiber length reduction enhancement agent is selected from the group consisting of: a hydrolyzed polymer or copolymer of vinylformamide with a degree of hydrolysis greater than 70% and a copolymer of acrylamide and acryloxyethyl-trimethylammonium chloride; and the reduction of papermaking fiber length of the modified pulp is from about 5% to about 20% and the pulp freeness level of the modified pulp is from about 300 CSF to about 650 CSF.
 42. The method of claim 34, wherein the papermaking fiber is a wood fiber and the fiber length reduction enhancement agent is selected from the group consisting of: a hydrolyzed polymer or copolymer of vinylformamide with a degree of hydrolysis greater than 70% and a copolymer of acrylamide and acryloxyethyl-trimethylammonium chloride; and is present at a dosage of from 0.5 kilogram per ton pulp (kg/ton pulp) to about 29 kg/ton pulp, wherein the pulp freeness level of the modified pulp is from about 350 CSF to about 650 CSF.
 43. The method of claim 34, wherein the fiber length reduction enhancement agent is selected from the group consisting of: a hydrolyzed polymer or copolymer of vinylformamide with a degree of hydrolysis greater than 70% and a copolymer of acrylamide and acryloxyethyl-trimethylammonium chloride; and is present at a dosage of from 0.5 kilogram agent per ton pulp (kg/ton pulp) to about 29 kg/ton pulp, and the reduction of papermaking fiber length of the modified pulp is from about 5% to about 20% and the pulp freeness level of the modified pulp is from about 300 CSF to about 650 CSF.
 44. An enzyme-modified fiber pulp according to claim
 34. 45. A method of modifying pulp fiber comprising contacting pulp comprising papermaking fibers with a thermostable or hyperthermostable cellulolytic enzyme at a temperature from about 70° C. to about 125° C., wherein the cellulase enzyme is substantially inactive at a temperature from about 35° C. to about 55° C.
 46. The method of claim 45, wherein the refining energy of the pulp or fibrous material is reduced by at least 10%.
 47. The method of claim 45, wherein the modified pulp has a hemicellulose content that is reduced by at least 0.2 wt. % compared to the hemicellulose content of a pulp made without contacting with the thermostable or hyperthermostable cellulolytic enzyme.
 48. The method of claim 45, wherein the cellulolytic enzyme is a cellulase, a cellulase-functioning hemicellulase enzyme, or a cellulase containing concomitant hemicellulase enzyme functions such as xylanases, mannanases, glucanases, and beta-glucanases.
 49. The method of claim 45, wherein the cellulolytic enzyme is a cellulase selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO:
 6. 50. The method of claim 45, wherein the contacting step further comprising a thermostable or hyperthermostable enzyme selected from the group consisting of: a hemicellulase, a mannanase, a xylanase, a pectinase, a laccase, a lignin peroxidase, a manganese peroxidase, and an oxidoreductase.
 51. The method of claim 45, wherein the pulp comprises papermaking fiber selected from the group consisting of: a softwood fiber, a hardwood fiber, and a mixture thereof.
 52. The method of claim 45, wherein the enzyme contacting step further comprises an intracrystalline lignocellulosic agent selected from the group consisting of an ionic liquid, an N-alkylated urea, an N-alkylated lactam, an N-alkylated amide, a polyol ether, a polyol, and combinations thereof.
 53. The method of claim 45, wherein the pulp comprises a kraft pulp, a sulfite pulp, a mechanical pulp, a semi-chemical pulp, a semi-mechanical pulp, a chemi-mechanical pulp, a fluff pulp, a tissue pulp, a dissolving pulp, a recycled pulp, a biorefinery pulp, or a deinked pulp.
 54. An enzyme-modified fiber pulp according to claim
 45. 55. A method of modifying papermaking pulp comprising: (1) contacting pulp comprising softwood fibers having a fiber length of about 2.1 mm to about 2.8 mm, as measured by FQA length-weighted fiber average length, with a cellulolytic enzyme at a temperature of about 35° C. to about 125° C., (2) refining the modified pulp, to produce a modified pulp having: (i) a fiber length of about 0.6 mm to about 1.7 mm, and (ii) a pulp freeness level of from about 400 Canadian Standard Freeness (CSF) to about 700 CSF.
 56. The method of claim 55, wherein the contacting step is carried out at a temperature of about 35° C. to about 125° C. for about 30 minutes to about 10 hours.
 57. The method of claim 55, wherein the contacting step is carried out at a temperature of about 35° C. to about 65° C. and at an enzyme dosage of from about 0.2 kilogram enzyme per ton of pulp (kg/T) to about 20 kg/T.
 58. The method of claim 57, wherein the cellulolytic enzyme is a cellulase selected from the group consisting of: a cellulase obtained or derived from Chrysosporium lucknowense/Myceliophthora thermophilia, a cellulase obtained or derived from Humicola insolens, a cellulase obtained or derived from Aspergillus, a cellulase obtained or derived from Trichoderma, and combinations thereof.
 59. The method of claim 55, wherein the contacting step is carried out at a temperature of about 66° C. to about 125° C. and at an enzyme dosage of from about 0.2 kilogram enzyme per ton of pulp (kg/T) to about 20 kg/T.
 60. The method of claim 55, wherein the cellulolytic enzyme is a cellulase or a cellulase-functioning hemicellulase enzyme.
 61. The method of claim 60, wherein the cellulolytic enzyme is a cellulase selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO:
 6. 62. The method of claim 55, further comprising contacting the pulp with a fiber length reduction enhancement agent.
 63. The method of claim 62, wherein contacting the pulp with the fiber length reduction enhancement agent contact occurs preferably after contacting the pulp with the enzyme.
 64. An enzyme-modified softwood fiber pulp having (i) a fiber length of about 0.6 mm to about 1.7 mm, and (ii) a pulp freeness level of from about 400 Canadian Standard Freeness (CSF) to about 700 CSF.
 65. A paper web or pulp product comprising a modified pulp according to claim
 64. 66. The paper web or pulp product of claim 65, wherein the product is selected from the group consisting of: printing writing papers, packaging papers, paper boards, personal care and hygiene products, paper tissues, paper towels, paper napkins, paper wipes, paper plates, paper cups, paper containers, deinked papers, recycled papers, microcrystalline cellulose, microfibrillated cellulose, nanofibrillated cellulose, nanocrystalline cellulose, dissolving pulp, and base papers for polymer impregnation or for composites.
 67. A paper web or pulp product comprising a modified pulp according to claim
 44. 68. A paper web or pulp product comprising a modified pulp according to claim
 54. 